THE  ELEMENTS  OF 

BACTERIOLOGICAL 
TECHNIQUE 

A  LABORATORY  GUIDE 


FOR  THE 


MEDICAL,  DENTAL,  AND   TECHNICAL  STUDENT 


BY 

J.  W.  H.  EYRE,  M.D.,  M.S.,  F.R.S.  EDIN. 

Bacteriologist  to  Guy's  Hospital,  London,  and  Lecturer  on  Bacteriology  in  the  Medical 

and  Dental  Schools  ;  formerly  Lecturer  on  Bacteriology  and  Joint-Lecturer 

on   Practical   Hygiene   in  the   Charing   Cross    Hospital  Medical 

School,  and  Bacteriologist  to  Charing  Cross  Hospital,  etc. 


WITH  170  ILLUSTRATIONS 


PHILADELPHIA  AND  LONDON 

W.    B.   SAUNDERS   &   COMPANY 
1903 


LIBRARY 


Copyright,  IQOZ,  by  W.  B.  Saunders  &  Company 


Registered  at  Stationers'  Hall,  London,  England 


TO  THE  MEMORY  OF 

JOHN  WICHENFORD  WASHBOURN,  C.M.G.,  M.D.,  F.R.C.P. 

Physician  to  Guy's  Hospital  and  Lecturer  on  Bacteriology  in  the 
Medical  School,  and  Physician  to  the  London  Fever  Hospital 

MY  TEACHER,  FRIEND,  AND  CO-WORKER 


225266 


PREFACE. 


IN  the  following  pages  I  have  endeavoured  to  arrange 
briefly  and  concisely  the  various  methods  at  present 
in  use  for  the  study  of  bacteria,  and  the  elucidation 
of  such  points  in  their  life-histories  as  are  debatable 
or  still  undetermined. 

Of  these  methods,  some  are  new,  others  are  not; 
but  all  are  .reliable,  only  such  having  been  included 
as  are  capable  of  giving  satisfactory  results  even  in 
the  hands  of  beginners.  In  fact,  the  bulk  of  the 
matter  is  simply  an  elaboration  of  the  typewritten 
notes  distributed  to  some  of  my  laboratory  classes  in 
practical  and  applied  bacteriology;  consequently  an 
attempt"  has  been  made  to  present  the  elements  of 
bacteriological  technique  in  their  logical  sequence. 

I  make  no  apology  for  the  space  devoted  to  illus- 
trations, nearly  all  of  which  have  been  prepared 
especially  for  this  volume;  for  a  picture,  if  good, 
possesses  a  higher  educational  value  and  conveys  a 
more  accurate  impression  than  a  page  of  print;  and 
even  sketches  of  apparatus  serve  a  distinct  purpose 
in  suggesting  to  the  student  those  alterations  and 
modifications  which  may  be  rendered  necessary  or 
advisable  by  the  character  of  his  laboratory  equip- 
ment. 

The  excellent  and  appropriate  terminology  intro- 
duced by  Chester  in  his  recent  work  on  "  Determina- 
tive Bacteriology"  I  have  adopted  in  its  entirety, 
for  I  consider  that  it  only  needs  to  be  used  to  convince 

one  of  its  extreme  utility,  whilst  its  inclusion  in  an 

ii 


1 2  PREFACE. 

elementary  manual  is  calculated  to  induce  in  the 
student  habits  of  accurate  observation  and  concise 
description. 

With  the  exception  of  Section  XVII,— "  Outlines 
for  the  Study  of  Pathogenic  Bacteria," — introduced 
with  the  idea  of  completing  the  volume  from  the  point 
of  view  of  the  medical  and  dental  student,  the  work 
has  been  arranged  to  allow  of  its  use  as  a  laboratory 
guide  by  the  technical  student  generally,  whether  of 
brewing,  dairying,  or  agriculture. 

So  alive  am  I  to  its  many  imperfections  that  it 
appears  almost  superfluous  to  state  that  the  book  is 
in  no  sense  intended  as  a  rival  to  the  many  and  ex- 
cellent manuals  of  bacteriology  at  present  in  use, 
but  aims  only  at  supplementing  the  usually  scanty 
details  of  technique,  and  at  instructing  the  student 
how  to  fit  up  and  adapt  apparatus  for  his  daily  work, 
and  how  to  carry  out  thoroughly  and  systematically 
the  various  bacterioscopical  analyses  that  are  daily 
demanded  of  the  bacteriologist  by  the  hygienist. 

Finally,  it  is  with  much  pleasure  that  I  acknowledge 
the  valuable  assistance  received  from  my  late  assistant, 
Mr.  J.  B.  Gall,  A.I.C.,  in  the  preparation  of  the  section 
dealing  with  the  chemical  products  of  bacterial  life, 
and  which  has  been  based  upon  the  work  of  Lehmann, 

JOHN  W.  H.  EYRE. 

GUY'S  HOSPITAL,  S.  E., 
September,  1902. 


CONTENTS. 


PAGE. 
I.  GLASS  APPARATUS  IN  COMMON  USE 17 

Cleaning  of  Glass  Apparatus,  24 — Plugging  Test-tubes  and 
Flasks,  29. 

II.  METHODS  OF  STERILISATION 32 

Sterilising  Agents,  32 — Methods  of  Application,  33. 

III.  THE  MICROSCOPE 51 

Methods  of  Micrometry,  60. 

IV.  MICROSCOPICAL  EXAMINATION  OF  BACTERIA  AND  OTHER  MI- 

CRO-FUNGI          65 

Apparatus  and  Reagents  Used  in  Ordinary  Microscopical 
Examination,  65 — Methods  of  Examination,  69. 

V.  STAINING  METHODS 81 

Bacteria  Stains,  81 — Contrast  Stains,  83 — Tissue  Stains,  84 
— Methods  of  Demonstrating  Structure  of  Bacteria,  86 — Dif- 
ferential Methods  of  Staining,  93. 

VI.  METHODS  OF  DEMONSTRATING  BACTERIA  IN  TISSUES    ....      98 
Freezing  Method,  99 — Paraffin  Method,  100 — Special  Stain- 
ing Methods  for  Sections,  104. 

VII.  CLASSIFICATION  OF  FUNGI        107 

Morphology  of  the  Hyphomycetes,  107 — Morphology  of  the 
Blastomycetes,  109. 

VIII.    SCHIZOMYCETES Ill 

Anatomy,  113 — Physiology,  Ii6 — Biochemistry,  123. 

IX.  NUTRIENT  MEDIA 125 

Meat  Extract,  127— Standardisation  of  Media,  132 — The 
Filtration  of  Media,  136 — Tubing  Nutrient  Mecfi&j-r^S. 

X.  STOCK  CULTURE  MEDIA 141 

XI.  INCUBATORS     .  < 174 

XII.  METHODS  OF  CULTIVATION 177 

Aerobic,  177 — Anaerobic  Cultivations,  186. 

XIII.  METHODS  OF  ISOLATION 196 

)(  XIV.  METHODS  OF  INDENTIFICATION       205 

Scheme  of  Study,  205 — Macroscopical  Examination  of 
Cultivations,  207 — Microscopical  Methods,  218 — Chemical 
Methods,  221 — Physical  Methods,  238. 

XV.  EXPERIMENTAL  INOCULATION  OF  ANIMALS 262 

Methods  of  Inoculation,  274. 

XVI.  POST-MORTEM  EXAMINATION  OF  EXPERIMENTAL  ANIMALS    .    .    287 

13 


14  CONTENTS. 

PAGE. 
XVII.  OUTLINES  FOR  THE  STUDY  OF  THE  PATHOGENIC  BACTERIA  .    .    294 

XVIII.  BACTERIOLOGICAL  ANALYSES     ........       .....    316 

Bacteriological  Examination  of  Water,  316 — Examination  of 
Sewage  and  Sewage  Effluents,  334 — Examination  of  Air,  335 
— Examination  of  Soil,  338 — Examination  of  Milk,  344 — 
Ice  Cream,  354 — Examination  of  Cream  and  Butter,  354 — 
Examination  of  Unsound  Meats,  356 — Examination  of  Fil- 
ters, 358 — Examination  of  Disinfectants,  359« 


INDEX ....    363 


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BACTERIOLOGICAL 
TECHNIQUE. 


L  GLASS  APPARATUS  IN  COMMON 
USE. 

THE  equipment  of  the  bacteriological  laboratory,  so 
far  as  the  glass  apparatus  is  concerned,  differs  but 
little  from  that  of  a  chemical  laboratory,  and  the  clean- 
liness of  the  apparatus  is  equally  important.  The 
glassware  comprised  in  the  following  list,  in  addition 
to  being  clean,  must  be  stored  in  a  sterile  or  germ- 
free  condition. 

Test=tubes. — It  is  convenient  to  keep  several  sizes 
of  test-tubes  in  stock,  to  meet  special  requirements, 
viz. : 

1.  1 8  by   1.5  cm.,  to  contain  media  for  ordinary 
tube  cultivations. 

2.  1 8  by  1.3  cm.,  to  contain  media  used  for  pouring 
plate  cultivations,  and  also  for  holding  sterile  "  swabs." 

3.  1 8  by  2  cm.,  to  contain  wedges  of  potato,  beet- 
root, or  other  vegetable  media. 

4.  13  by  1.5  cm.,  to  contain  inspissated  blood-serum. 
The  tubes  should  be  made  from  the  best  German 

potash  glass,  "  blue-lined,"  stout  and  heavy,  with  the 
edge  of  the  mouth  of  the  tube  slightly  turned  over, 
but  not  to  such  an  extent  as  to  form  a  definite  rim. 
(Cost  about  $4.25,  or  9  shillings  per  gross.)  Such 
tubes  are  sufficiently  stout  to  resist  rough  handling, 
do  not  usually  break  if  accidentally  allowed  to  drop, 

can  be  cleaned,  sterilised,  and  used  over  and  over  again, 
2  17 


1 8  GIvASS   APPARATUS    IN   COMMON    USE. 

and  by  their  length  of  life  fully  justify  their  initial 
expense. 

A  point  to  be  noted  is  that  the  manufacturers  rarely 
turn  out  such  tubes  as  these  absolutely  uniform  in 
calibre,  and  a  batch  of  1 8  by  1.5  cm.  tubes  usually  con- 
tains such  extreme  sizes  as  1 8  by  2  cm.  and  18  by  1.3 
cm.  Consequently,  if  a  set  of  standard  tubes  is  kept 
for  comparison,  each  new  supply  of  1 8  by  1.5  cm.  tubes 
may  be  easily  sorted  out  into  these  three  sizes,  and  so 
simplify  ordering. 

5.  5  by  0.9  cm.,  for  use  in  the  inverted  position 
inside  the  tubes  containing  carbohydrate  media,  as 
gas-collecting  tubes. 

6.  5  by  0.5  cm.,  for  sedimentation  reactions,  etc. 


Fig.  I. — Bohemian  flask.      Fig.  2. — Pear-shaped     Fig.  3. — Erlenmeyer  flask 

flask.  (narrow  neck). 

These  tubes  may  be  of  common  thin  glass,  "un- 
rimmed,"  as  less  than  two  per  cent,  are  fit  for  use  a 
second  time. 

Bohemian  Flasks  (Fig.  i). — These  are  the  ordinary 
flasks  of  the  chemical  laboratory.  A  good  variety, 
ranging  in  capacity  from  250  to  3000  c.c.,  should  be 
kept  on  hand.  A  modified  form,  known  as  the  "  pear- 
shaped"  (Fig.  2),  is  preferable  f6r  the  smaller  sizes— 
i.  e.,  250  and  500  c.c. 

Erlenmeyer's  Flasks  (Fig.  3). — Erlenmeyer's  flasks 
of  75,  loo,  and  250  c.c.  capacity  are  extremely  useful. 
For  use  as  culture  flasks  care  should  be  taken  to  select 


PETRl'S   DISHES   OR    "PIRATES." 


only  such  as  have  a  narrow  neck  of  about  2  cm.  in 
length. 

Kolle's  Culture  Flasks  (Fig.  4).— These  thin,  flat 
flasks  (to  contain  agar  or  gelatine,  which  is  allowed  to 
solidify  in  a  layer  on  one  side)  are  extremely  useful 
on  account  of  the  large  nutrient  surface  available  for 
growth.  A  surface  cultivation  in  one  of  these  will 
yield  as  much  growth  as  ten  or  twelve  "  oblique"  tube 
cultures.  The  wide  mouth,  however,  is  a  disadvantage, 
and  for  many  purposes  thin,  flat  culture  bottles  (Fig.  5) 
are  to  be  preferred. 

Filter  Flasks  or  Kitasato's  Serum  Flasks  (Fig.  6).— 
Various  sizes,  from  250  to  2000  c.c.  capacity.  These 


Fig.  4.— Kolle  flask.      Fig.  5.— Culture  bottles.      Fig.  6.— Filter  flask. 

must  be  of  stout  glass,  to  resist  the  pressure  to  which 
they  are  subjected,  and  must  be  thoroughly  well  an- 
nealed, in  order  to  withstand  the  temperature  neces- 
sary for  sterilisation. 

Petri's  Dishes  or  "Plates"  (Fig.  7,  a).— These  have 
now  completely  replaced  the  rectangular  sheets  of  glass 
introduced  by  Koch  for  the  plate  method  of  cultiva- 
tion. Each  "plate"  consists  of  a  pair  of  circular  discs 
of  glass  with  sharply  upturned  edges,  thus  forming 
shallow  dishes,  one  of  slightly  greater  diameter  than 
the  other,  and  so,  when  inverted,  forming  a  cover  or 


2O 


GLASS    APPARATUS   IN    COMMON   USE. 


cap  for  the  smaller.  Plates  having  an  outside  diam- 
eter of  10  cm.  and  a  height  of  1.5  cm.  are  the  most 
generally  useful.  Such  plates  are  sterilised  and  stored 
in  batches  of  eighteen  in  cylindrical  copper  boxes  with 
a  "pull-off"  lid,  30  cm.  high  by  12  cm.  diameter.  In- 
side the  box  is  a  copper  stirrup  with  a  circular  bottom, 
upon  which  the  plates  rest,  and  by  means  of  which 
each  can  be  raised  in  turn  to  the  mouth  of  the  box 
(Fig.  8). 

Capsules  (Fig.  7,  b  and  c). — These  are  Petri's  dishes 
of  smaller  diameter  but  greater  depth  than  those  termed 


Fig.  7. — Petri  dish  and  capsules. 


Fig.  8.-— Plate  box  with 
stirrup. 


plates.  Two  sizes  will  be  found  useful — viz.,  4  cm. 
diameter  by  2  cm.  high,  capacity  about  14  c.c.;  and  5 
cm.  diameter  by  2  cm.  high,  capacity  about  25  c.c. 
These  are  stored  in  copper  cylinders  of  similar  con- 
struction to  those  used  for  plates,  but  measuring  20 
by  6  cm.  and  20  by  7  cm.,  respectively. 

Graduated  Pipettes. — Several  varieties  of  these  are 
required,  viz. : 

1.  Pipettes  of  i  c.c.  capacity  graduated  in  o.i  c.c. 

2.  Pipettes  of  i  c.c.  capacity  graduated  in  o.oi  c.c. 
(Fig.  9,  a). 


CAPILLARY  PIPETTES   OR   PASTEUR'S   PIPETTES.     21 


3.  Pipettes  of  10  c.c.  capacity  graduated  in  o.i  c.c. 

(Fig.  9,*). 

These  should  be  about  30  cm.  in  length  (i  and  2 
of  fairly  narrow  bore),  graduated  to  the  extreme  point, 
and  having  at  least  a  10  cm.  length  of  clear  space  be- 
tween the  first  graduation  and  the  upper  end.  Each 
variety  should  be  stored  in  a  separate  cylindrical  copper 
case  some  36  by  6  cm.,  with  "  pull-off"  lid,  upon  which 
is  stamped,  in  plain  figures,  the  capacity  of  the  con- 
tained pipettes. 


V  U 

a  b 

Fig.  9. — Measuring  pipettes,  a  and  b. 


a  b  c 

Fig.  10. — Pasteur's  pipettes,  a,  b,  c. 


Capillary  Pipettes  or  Pasteur's  Pipettes  (Fig.  10,  a). — 
These  little  instruments  are  invaluable,  and  a  goodly 
supply  should  be  kept  on  hand.  They  are  prepared 
from  soft  glass  tubing  of  various-sized  calibre  in  the 
following  manner:  Hold  a  14  cm.  length  of  glass  tube 
by  each  end,  and  whilst  rotating  it  heat  the  central 
portion  in  the  Bunsen  flame  until  the  glass  is  red  hot 
and  soft.  Now  remove  it  from  the  flame  and  steadily 
pull  the  ends  apart,  so  drawing  the  heated  portion 
out  into  a  capillary  tube;  break  the  capillary  portion 


22 


GLASS    APPARATUS    IN    COMMON    USE. 


at  its  centre,  seal  the  broken  ends  in  the  flame,  and 
round  off  the  edges  of  the  open  end  of  each  pipette. 
A  loose  plug  of  cotton-wool  in  the  open  mouth  com- 
pletes the  capillary  pipette.  After  a  number  have  been 
prepared,  they  are  sterilised  and  stored  in  batches, 
either  in  metal  cases  similar  to  those  used  for  the  gradu- 
ated pipettes  or  in  large-sized  test-tubes — sealed  ends 
downwards  and  plugged  ends  towards 
the  mouth  of  the  case. 

A  modification  of  this  pipette,  in 
which  a  constriction  or  short  length 
of  capillary  tube  is  introduced  just 
below  the  plugged  mouth  (Fig.  10,  b), 
will  also  be  found  extremely  useful  in 
the  collection  and  storage  of  morbid 
exudations. 

A  third  form,  where  the  capillary 
portion  is  about  4  or  5  cm.  long  and 
only  forms  a  small  fraction  of  the 
entire  length  of  the  pipette  (Fig.  10, 
c),  will  also  be  found  useful. 

"Blood"  Pipettes  (Fig.  n).— Spe- 
cial pipettes  for  the  collection  of  fairly 
large  quantities  of  blood  (as  suggested 
by  Pakes)  should  also  be  prepared. 
These  are  made  from  soft  glass  tub- 
ing of  i  cm.  bore  in  a  similar  manner 
to  the  Pasteur  pipettes,  except  that 
a  blowpipe  flame  must  be  used  in  order  to  obtain  the 
sharp  shoulder  at  either  end  of  the  central  bulb.  The 
terminal  tubes  must  retain  a  diameter  of  at  least  i 
mm.,  in  order  to  avoid  capillary  action  during  the  col- 
lection of  the  fluid. 

For  sterilisation  and  storage  each  pipette  is  placed 
inside  a  test-tube,  resting  on  a  wad  of  cotton-wool, 
and  the  tube  plugged  in  the  ordinary  manner.  As 
these  tubes  are  used  almost  exclusively  for  blood  work, 


Fig.  II.  — Blood 
pipettes  and  hare-lip 
pin  in  a  test-tube. 


FERMENTATION   TUBES. 


it  is  usual  to  place  a  lance-headed  hare-lip  pin  inside 
the  tube  so  that  the  entire  outfit  may  be  sterilised  at 
one  time. 

Graduated  Capillary  Pipettes  (Fig.  12). — These  should 
also  be  made  in  the  laboratory, — from  manometer 
tubing, — of  simple,  convenient  shape, 
and  graduated  by  the  aid  of  a  i  c.c. 
pipette  (in  hundredths)  to  contain 
such  quantities  as  10,  50,  and  90 
c.mm.,  and  carefully  marked  with  a 
writing  diamond.  These,  previously 
sterilised  in  large  test-tubes,  will  be 
found  extremely  useful  in  preparing 
accurate  percentage  solutions,  when 
only  minute  quantities  of  fluid  are 
available. 

Sedimentation  Tubes  (Fig.  13).— 
These  are  prepared  from  10  cm. 
lengths  of  narrow  glass  tubing  by 
sealing  one  extremity,  blowing  a 
small  bulb  at  the  centre,  and  plugging  the  open  end 
with  cotton- wool;  after  sterilisation  the  open  end  is 
provided  with  a  short  piece  of  rubber  tubing  and  a  glass 
mouthpiece.  When  it  is  necessary  to  observe  sedi- 
mentation reactions  in  very  small  quantities  of  fluid, 


Fig.    12.  —  Capillary 
graduated  pipettes. 


Fig.  13. — Sedimentation  tube. 

these  tubes  will  be  found  much  more  convenient  than 
the  5  by  0.5  cm.  test-tubes  previously  mentioned. 

Fermentation  Tubes  (Fig.  14). — These  are  used  for 
the  collection  and  analysis  of  the  gases  liberated  from 
the  media  during  the  growth  of  some  varieties  of  bac- 


GLASS    APPARATUS    IN    COMMON    USE. 


teria  and  may  be  either  plain  (a)  or  graduated  (6). 
A  simple  form  (Fig.  14,  c)  may  be  made  from  14 
cm.  lengths  of  soft  glass  tubing  of  1.5  cm.  diameter. 
The  Bunsen  flame  is  applied  to  a  spot  some  5  cm.  from 
one  end  of  such  a  piece  of  tubing  and  the  tube  slightly 
drawn  out  to  form  a  constriction,  the  constricted  part 


Fig.  14. — Fermentation  tubes. 

is  bent  in  the  bat's-wing  flame,  to  an  acute  angle,  and 
the  open  extremity  of  the  long  arm  sealed  off  in  the 
blowpipe  flame.  The  open  end  of  the  short  arm  is 
rounded  off  and  then  plugged  with  cotton- wool,  and 
the  tube  is  ready  for  sterilisation. 

CLEANING  OF  GLASS  APPARATUS. 

All  glassware  used  in  the  bacteriological  laboratory 
must  be  thoroughly  cleaned  before  use,  and  this  rule 
applies  as  forcibly  to  new  as  to  old  apparatus,  although 
the  methods  employed  may  vary  slightly. 

To  Clean  New  Test=tubes.— 

1.  Place  the  tubes  in  a  bucket  or  other  convenient 
receptacle,  fill  with  water  and  add  a  handful  of  "  sapon" 
or  other  soap  powder.     See  that  the  tubes  are  full  and 
submerged. 

2.  Fix  the  bucket  over  a  large  Bunsen  flame  and  boil 
for  thirty  minutes. 

3.  Cleanse  the  interior  of  the  tubes  with  the  aid  of 
test-tube  brushes,  and  rinse  thoroughly  in  cold  water. 


INFECTED   TEST-TUBES.  25 

4.  Invert  the  tubes  and  allow  them  to  drain  com- 
pletely. 

5.  Dry  the  tubes  and  polish  the  glass  inside  and  out 
with  a  soft  cloth,  such  as  selvyt. 

New  flasks,  plates,  and  capsules  must  be  cleaned  in 
a  similar  manner. 

To  Clean  New  Graduated  Pipettes.— 

1.  Place  the  pipettes  in  a  convenient  receptacle, 
filled  with  water  to  which  soap  powder  has  been  added. 

2.  Boil  the  water  vigorously  for  twenty  minutes  over 
a  Bunsen  flame. 

3.  Rinse  the  pipettes  in  running  water  and  drain. 

4.  Run   distilled   water   through   the   pipettes   and 
drain. 

5.  Run  rectified  spirits  through  the  pipette  and  drain 
as  completely  as  possible. 

6.  Place  the  pipettes  in  the  hot-air  oven  (vide  page 
35),  close  the  door,  open  the  ventilating  slide,  and 
run  the  temperature  slowly  up  to  about  80°  C.     Turn 
off  the  gas  and  allow  the  oven  to  cool. 

Or  6a.  Attach  each  pipette  in  turn  to  the  rubber 
tube  of  the  foot  bellows,  or  blowpipe  air-blast,  and 
blow  air  through  the  pipette  until  the  interior  is  dry. 

Glassware  that  has  already  been  used  is  regarded  as 
infected,  and  is  treated  in  a  slightly  different  manner. 

Infected  Test=tubes.— 

1.  Pack  the  tubes  in  the  wire  basket  of  the  auto- 
clave   (having   previously   removed    the    cotton-wool 
plugs,  caps,  etc.),  in  the  vertical  position,  and  before 
replacing  the  basket  see  that  there  is  a  sufficiency  of 
water  in  the  bottom  of  the  boiler.     Now  attach  a 
piece  of  rubber  tubing  to  the  nearest  water  tap,  and 
by  means  of  this  fill  each  tube  with  water. 

2.  Disinfect  completely  by  exposing  the  tubes,  etc., 
to  a  temperature  of  120°  C.  for  twenty  minutes  (vide 
page  42). 

(If  an  autoclave  is  not  available,  the  tubes  must  be 


26  GLASS    APPARATUS    IN    COMMON   USE. 

placed  in  a  digester,  or  even  a  large  pan  or  pail  with  a 
tightly  fitting  cover,  and  boiled  vigourously  for  some 
thirty  to  forty-five  minutes  to  ensure  disinfection.) 

3.  Whilst  still  hot,  empty  each  tube  in  turn  and 
roughly  clean  its  interior  with  a  stiff  test-tube  brush. 

4.  Place  the  tubes  in  a  bucket  or  other  convenient 
receptacle,  fill  with  water  and  add  a  handful  of  sapon 
or  other  soap  powder.     See  that  the  tubes  are  full  and 
submerged. 

5.  Fix  the  bucket  over  a  large  Bunsen  flame  and 
boil  for  thirty  minutes. 

6.  Cleanse  the  interior  of  the  tubes  with  the  aid  of 
test-tube  brushes,  and  rinse  thoroughly  in  cold  water. 

7.  Drain  off  the  water  and  immerse  tubes  in  a  large 
jar  containing  water  acidulated  with  2  to  5  per  cent, 
hydrochloric  acid.     Allow  them  to  remain  there  for 
about  fifteen  minutes. 

8.  Remove  from  the  acid  jar,  drain,  rinse  thoroughly 
in  running  water,  then  with  distilled  water. 

9.  Invert  the  tubes  and  allow  them  to  drain  com- 
pletely. 

Dry  the  tubes  and  polish  the  glass  inside  and  out 
with  a  soft  cloth,  such  as  selvyt, 

Infected  flasks,  plates,  and  capsules  must  be  treated 
in  a  similar  manner. 

Flasks  which  have  been  used  only  in  the  preparation 
of  media  must  be  cleaned  immediately  they  are  finished 
with.  Fill  each  flask  with  water  to  which  some  soap 
powder  and  a  few  crystals  of  potassium  permanganate 
have  been  added,  and  let  boil  over  the  naked  flame. 
The  interior  of  the  flask  can  then  usually  be  perfectly 
cleaned  with  the  aid  of  a  flask  brush,  but  in  some  cases 
water  acidulated  with  5  per  cent,  nitric  acid,  or  a  large 
wad  of  wet  cotton- wool  previously  rolled  in  silver  sand, 
must  be  shaken  around  the  interior  of  the  flask,  after 
which  rinse  thoroughly  with  clean  water,  dry,  and 
polish. 


COVER-SUPS.  27 

Infected   Pipettes.— 

1.  Plunge  infected  pipettes  immediately  after  use 
into  tall  glass  cylinders  containing  a  2  per  cent,  solu- 
tion of  lysol,  and  allow  them  to  remain  therein  for 
some  days. 

2.  Remove  from  the  jar  and  drain.     Boil  in  water 
to  which  a  little  soap  has  been  added,  for  thirty  min- 
utes. 

3.  Rinse  thoroughly  in  cold  water. 

4.  Immerse  in  5  per  cent,  nitric  acid  for  an  hour  or 
two. 

5.  Rinse  again  in  running  water  to  remove  all  traces 
of  acid. 

6.  Complete  the  cleaning  as  described  under  "new 
pipettes." 


*ft 

x 

<9m*i. 

ZZ™™" 

/* 

X 

w     t 

n. 

|  iQmm': 

«T 

i 

I 

Fig.  15. — Slides  and  cover-slips,  actual  size. 

Slides  and  cover=slips  (Fig.  15),  when  first  purchased, 
have  "  greasy"  surfaces,  upon  which  water  gathers  in 
minute  drops  and  effectually  prevents  the  spreading 
of  thin,  even  films. 

Microscopical  Slides. — The  slides  in  general  use  are 
those  known  as  "three  by  one"  slips  (measuring  3 
inches  by  i  inch,  or  76  by  26  mm.),  and  should  be 
of  good  white  crown  glass,  with  ground  edges. 

New  slides  should  be  allowed  to  remain  in  alcohol 
acidulated  with  5  per  cent,  hydrochloric  acid  for  some 
hours,  rinsed  in  running  water,  roughly  drained  on  a 
towel,  dried,  and  finally  polished  with  a  selvyt  cloth. 

Cover=slips. — The  most  useful  sizes  are  the  19  mm. 


28  GLASS   APPARATUS   IN    COMMON   USE. 

squares  for  ordinary  cover-glass  film  preparations,  and 
38  by  19  mm.  rectangles  for  blood  films  and  serial  sec- 
tions; both  varieties  must  be  of  "No.  i"  thickness, 
which  varies  between  0.15  and  0.22  mm.,  that  they 
may  be  available  for  use  with  the  high-power  immer- 
sion lenses. 

Cover-slips  should  be  cleaned  in  the  following  manner: 

1 .  Drop  the  cover-slips  one  by  one  into  an  enamelled 
iron  pot  or  tall  glass  beaker,  containing  a  10  per  cent, 
solution  of  chromic  acid. 

2.  Heat  over  a  Bunsen  flame  and  allow  the  acid 
to  boil  gently  for  twenty  minutes. 

NOTE. — A  few  pieces  of  pipe-clay  or  pumice  may  be 
placed  in  the  beaker  to  prevent  the  "spurting"  of  the 
chromic  acid. 

3.  Turn  the  cover- slips  out  into  a  flat  glass  dish  and 
wash  in  running  water  under  the  tap  until  all  trace 
of  yellow  colour  has  disappeared.     During  the  wash- 
ing keep  the  cover-slips  in  motion  by  imparting  a 
rotatory  movement  to  the  dish. 

4.  Wash  in  distilled  water  in  a  similar  manner. 

5.  Wash  in  rectified  spirit. 

6.  Transfer  the  cover-slips,  by  means  of  a  pair  of 
clean  forceps,  previously  heated  in  the  Bunsen  flame 
to  destroy  any  trace  of  grease,  to  a  small  beaker  of 
absolute  alcohol. 

Drain  off  the  alcohol  and  transfer  the  cover-slips, 
by  means  of  the  forceps,  to  a  wide-mouthed  glass  pot, 
containing  absolute  alcohol,  in  which  they  are  to  be 
stored,  and  stopper  tightly. 

NOTE. — After  once  being  placed  in  the  chromic  acid, 
the  cover-slips  must  on  no  account  be  touched  by  the 
fingers. 

Used  Slides  and  Cover=slips. — Used  slides  with  the 
mounted  cover-slip  preparations,  and  cover-slips  used 
for  hanging-drop  mounts,  should,  when  discarded,  be 


PLUGGING  TEST-TUBES  AND  FLASKS.  29 

thrown  into  a  pot  containing  a  2  per  cent,  solution  of 
lysol. 

After  immersion  therein  for  a  week  or  so,  even  the 
cover-slips  mounted  with  Canada  balsam  can  be  readily 
detached  from  their  slides. 

Slides. — 

1 .  Wash  the  slides  thoroughly  in  running  water. 

2.  Boil  the  slides  in  water  to  which  "sapon"  has 
been  added,  for  half  an  hour. 

3.  Rinse  thoroughly  in  cold  water. 

4.  Dry  and  polish  with  a  dry  cloth. 
Cover-slips.— 

1 .  Wash  the  cover-slips  thoroughly  in  running  water. 

2.  Boil  the  cover- slips  in   10  per  cent,  solution  of 
chromic  acid,  as  for  new  cover-slips. 

3.  Wash  thoroughly  in  running  water. 

4.  Pick  out  those  cover-slips  which  show  much  ad- 
herent dirty  matter,  and  rub  them  between  thumb 
and  forefinger  under  the  water  tap.     The  dirt  usually 
rubs  off  easily,  as  it  has  become  friable  from  contact 
with  the  chromic  acid. 

5.  Return  all  the  cover-slips  to  the  beaker,  fill  in 
fresh  chromic  acid  solution,  and  treat  as  new  cover- 
slips. 

NOTE. — Test-tubes,  plates,  capsules,  etc.,  which,  from 
long  use,  have  become  scratched  and  hazy,  or  which 
cannot  be  cleaned  in  any  other  way,  may  be  dealt 
with  by  immersing  them  in  an  enamelled  iron  bath, 
containing  water  acidulated  with  hydrofluoric  acid  i 
per  cent.,  for  ten  minutes,  rinsing  thoroughly  in  water, 
drying,  and  polishing. 

PLUGGING  TEST-TUBES  AND  FLASKS. 

Before  sterilisation  all  test-tubes  and  flasks  must 
be  carefully  plugged  with  cotton- wool,  and  for  this 
purpose  best  absorbent  cotton-wool  (preferably  that 
put  up  in  cylindrical  one-pound  packets  and  inter- 


3O  GIvASS    APPARATUS    IN   COMMON   USE. 

leaved  with  tissue  paper — known  as  surgeons'  wool) 
should  be  employed. 

1.  For  a  test-tube  or  a  small  flask,  tear  a  strip  of 
cotton- wool  some  10  cm.  long  by  2  cm.  wide  from  the 
roll. 

2.  Turn  in  the  ends  neatly  and  roll  the  strip  of  wool 
lightly  between  the  thumb  and  fingers  of  both  hands 
to  form  a  long  cylinder. 

3.  Double  this  at  the  centre  and  introduce  the  now 
rounded  end  into  the  open  mouth  of  the  tube  or  flask. 


Fig.  16. — Plugging  test-tubes  :  a,  Cylinder  of  wool  being  rolled;  b,  cylinder 
of  wool  being  doubled ;  r,  cylinder  of  wool  being  inserted  in  tube. 

4.  Now,  whilst  supporting  the  wool  between  the 
thumb  and  fingers  of  the  right  hand,  rotate  the  test- 
tube  between  those  of  the  left,  and  gradually  screw 
the  plug  of  wool  into  its  mouth  for  a  distance  of  about 
2.5  cm.,  leaving  about  the  same  length  of  wool  project- 
ing. 

The  plug  must  be  firm  and  fit  the  tube  or  flask  fairly 
tightly,  sufficiently  tightly  in  fact  to  bear  the  weight 
of  the  glass  plus  the  amount  of  medium  the  vessel  is 
intended  to  contain,  but  not  so  tightly  as  to  prevent  it 
from  being  easily  removed  by  a  screwing  motion  when 


PLUGGING  TEST-TUBES  AND  FLASKS.  31 

grasped  between  the  fourth,  or  third  and  fourth  fingers, 
and  the  palm  of  the  hand. 

For  a  large  flask  a  similar  but  larger  strip  of  wool 
must  be  taken;  the  method  of  making  and  inserting 
the  plug  is  identical 


IL  METHODS  OF  STERILISATION. 

STERILISING  AGENTS. 

STERILISATION — i.  e.,  the  removal  or  the  destruction 
of  germ  life — may  be  effected  by  the  aid  of  various 
agents.  As  applied  to  the  requirements  of  the  bac- 
teriological laboratory,  many  of  these  agents,  such  as 
electricity,  sunlight,  etc.,  are  practically  useless ;  whilst 
others  are  limited  in  their  applications,  or  are  so  well 
suited  to  particular  purposes  as  to  be  almost  entirely 
restricted  to  such. 

The  sterilising  agents  in  common  use  are: 

Chemical  Reagents. — Disinfectants  (for  the  disinfec- 
tion of  glass  and  metal  apparatus  and  of  morbid  tis- 
sues). 

Heat.— (a)  Dry  Heat: 

1.  Naked  flame   (for  the  sterilisation  of  platinum 
needles,  etc.). 

2.  Muffle  furnace  (for  the  sterilisation  of  filter  can- 
dles, and  for  the  destruction  of  morbid  tissues). 

3.  Hot  air  (for  the  sterilisation  of  all  glassware  and 
of  metallic  substances). 

(b)  Moist  Heat: 

1.  Water  at  56°  C.  (for  the  sterilisation  of  certain 
albuminous  fluids). 

2.  Water  at  100°  C.  (for  the  sterilisation  of  surgical 
instruments,  rubber  tubing,  and  stoppers,  etc.). 

3.  Streaming  steam  at  100°  C.  (for  the  sterilisation 
of  media). 

4.  Superheated  steam  at  115°  C.  or  120°  C.  (for  the 
disinfection  of  contaminated  articles  and  old  culti- 
vations of  bacteria). 

32 


METHODS    OF   APPLICATION.  33 

Filters.- 

1 .  Cotton- wool  filters  (for  the  sterilisation  of  air  and 
gases). 

2.  Porcelain  filters  (for  the  sterilisation  of  various 
liquids). 

METHODS  OF  APPLICATION. 

Chemical  Reagents,  such  as  belong  to  the  class  known 
as  antiseptics  (i.  e.,  substances  which  inhibit  the  growth 
of,  but  do  not  destroy,  bacterial  life),  are  obviously 
useless.  Disinfectants  or  germicides  (i.  e.,  substances 
which  destroy  bacterial  life),  on  the  other  hand,  are  of 
value  in  the  disinfection  of  morbid  material,  and  also 
of  various  pieces  of  apparatus,  such  as  pipettes,  pend- 
ing their  cleansing  and  complete  sterilisation  by  other 
processes.  To  this  class  belong: 

1.  Lysol,  2  per  cent,  solution; 

2.  Perchloride  of  mercury,  o.i  per  cent,  solution; 

3.  Carbolic  acid,  5  per  cent,  solution; 

4.  Absolute  alcohol; 
5-  Ether; 

6.  Chloroform; 

7.  Volatile  oils,  such  as  oil  of  mustard,  oil  of  garlic, 
arranged  in  order  of  general  utility.     Formaldehyde  is 
perhaps  a  more  powerful  germicide  than  any  of  the 
above,  but  its  penetrating  vapor  restricts  its  use.  These 
disinfectants  are  but  little  used  in  the  final  sterilisa- 
tion of  apparatus,  chiefly  on  account  of  the  difficulty 
of   completely   removing   such   substances,   for   even 
traces  of  these  chemicals  are  sufficient  to  so  inhibit 
or  alter  the  growth  of  bacteria  brought  into  contact 
with  them,  as  to  vitiate  subsequent  experiments  con- 
ducted by  the  aid  of  apparatus  sterilised  in  this  manner. 

NOTE. — Tubes,   flasks,   filter  flasks,   pipettes,  glass 
tubing,  etc.,  may  be  rapidly  sterilised,  in  case  of  emer- 
gency, by  washing,  in  turn,  with  distilled  water,  per- 
chloride  of  mercury  solution,  alcohol,  and  ether,  drain- 
3 


34 


METHODS    OF    STERILISATION. 


ing,  and  finally  gently  heating  over  a  gas  flame  to  com- 
pletely drive  off  the  ether  vapor.  Chloroform  or  vola- 
tile oils  may  be  added  to  various  fluids  in  order  to 
effect  the  destruction  of  contained  bacteria,  and  when 
this  has  been  done,  may  be  completely  driven  off  from 
the  fluid  by  the  application  of  gentle  heat. 

Dry  Heat. — The  naked  flame  of  the  Bunsen  burner 
is  used  for  sterilising  the  platinum  needles,  the  points 
of  forceps,  or  other  small  instruments,  cover-glasses, 
etc.,  a  very  short  exposure  to  this  heat  being  sufficient 
to  ensure  sterilisation. 

Muffle  Furnace    (Fig.    17). — This  form  of  heat  is 

chiefly  used  for  the  destruc- 
tion of  the  dead  bodies  of 
small  infected  animals,  mor- 
bid tissues,  etc.,  but  is  also 
employed  for  the  sterilisation 
of  porcelain  filter  candles. 

Filter  candles  are  disin- 
fected immediately  after  use 
by  boiling  in  a  beaker  of 
water  for  some  fifteen  or 
twenty  minutes.  This  treat- 
ment, however,  leaves  the 
dead  bodies  of  the  bacteria 

upon  the  surface  and  blocking  the  interstices  of  the 
filter. 

To  destroy  the  organic  matter  and  prepare  the  filter 
candle  for  further  use  proceed  as  follows : 

1.  Roll  each  bougie  up  in  a  piece  of  asbestos  cloth, 
secure  the  ends  with  a  few  turns  of  copper  wire,  and 
place  inside  the  muffle  (a  small  muffle  76  by  88  by 
163  mm.  will  hold  perhaps  four  small  Berkfeld  candles). 

2.  Light  the  gas  and  raise  the  contents  of  the  muffle 
to  a  white  heat;  maintain  this  temperature  for  five 
minutes. 

3.  Extinguish  the  gas,  and  when  the  muffle  and  its 


Fig.  17. — Muffle  furnace. 


HOT    AIR. 


35 


contents  have  become  quite  cold,  remove  the  filter 
candles  from  the  interior  of  the  muffle,  and  store  with- 
out removing  the  asbestos  wrappings,  in  sterile  metal 
boxes. 

Hot  Air. — Hot  air  at  150°  C.  destroys  all  bacteria, 
spores,  etc.,  in  about  thirty  minutes,  whilst  a  few 
minutes'  exposure  to  a  temperature  of  170°  to  180° 
C.  will  effect  the  same  result.  This  method  is  only 
applicable  to  glass  and  metallic  substances,  and 
the  small  bulk  of  cot- 
ton-wool comprised  in 
the  test-tube  plugs, 
etc.  Large  masses  of 
fabric  are  not  effectu- 
ally sterilised  by  dry 
heat,  as  its  power  of 
penetration  is  not 
great. 

Sterilisation  by  hot 
air  is  effected  in  the 
hot-air  oven  (Fig.  18). 
This  is  a  rectangular, 
double -walled  metal 
box,  mounted  on  a 
stand  and  heated  from 


Fig.  1 8. — Hot-air  oven. 


below  by  a  large  Bun- 
sen   burner.      One   of 

the  sides  is  hinged  to  form  a  door.  The  central  portion 
of  the  metal  bottom,  on  which  the  Bunsen  flame  would 
play,  is  cut  away,  and  replaced  by  firebrick  plates, which 
slide  in  metal  grooves  and  are  easily  replaced  when 
broken  or  worn  out.  The  top  of  the  oven  is  provided 
with  a  perforated  ventilator  slide  and  two  tubulures, 
the  one  for  the  reception  of  a  centigrade  thermometer 
graduated  to  200°  or  250°  C.,  the  other  for  a  thermo- 
regulator.  The  thermo-regulator  is  by  no  means  a 
necessity,  and  it  is  more  convenient  to  replace  it  by  a 


36  METHODS    OF   STERILISATION. 

large  bore  thermometer  with  a  sliding  platinum  point, 
connected  with  an  electric  bell,  which  can  be  easily 
adjusted  to  ring  at  any  given  temperature.  The  in- 
terior of  the  oven  is  provided  with  loose  shelves  upon 
which  the  articles  to  be  sterilised  are  arranged,  either 
singly  or  packed  in  square  wire  baskets  or  crates,  kept 
specially  for  this  purpose. 

To  prepare  crates  for  the  reception  of  test-tubes,  etc., 
cover  the  bottom  with  a  layer  of  thick  asbestos  cloth ;  or 
take  some  asbestos  fibre,  moisten  it  with  a  little  water  and 
knead  it  into  a  paste ;  plaster  the  paste  over  the  bottom  of 
each  crate,  working  it  into  meshes  and  smoothing  its  surface 
by  means  of  a  pestle.  When  several  crates  have  been  thus 
treated,  place  them  inside  the  hot-air  oven,  close  the  door, 
open  the  ventilating  slide,  light  the  gas,  and  run  the  temper- 
ature of  the  interior  up  to  160°  C.  After  an  interval  of 
about  ten  minutes  extinguish  the  gas,  open  the  oven  door, 
and  allow  the  contents  to  cool.  The  asbestos  now  forms 
a  smooth,  dry,  spongy  layer  over  the  bottom,  which  will 
last  many  months  before  needing  renewal,  and  will  con- 
siderably diminish  the  loss  of  tubes  from  breakage. 

Copper  cylinders  and  large  test-tubes  intended  for  the 
reception  of  pipettes  are  prepared  in  a  similar  manner,  to 
protect  the  points  of  these  articles. 

To  USE  THE  HOT-AIR  OVEN. — 

1 .  Place  the  crates  of  test-tubes,  plates  and  pipettes 
in  their  metal  cases,  loose  apparatus,  etc.,  inside  the 
oven,  taking  particular  care  that  none  of'  the  cotton- 
wool plugs  are  in  contact  with  the  walls  of  the  oven, 
otherwise  the  heat  transmitted  by  the  metal  will  char 
or  even  flame  them. 

2.  Close  the  oven  door,  set  the  electric  alarm  to  ring 
at  100°  C.,  light  the  gas  below,  and  open  the  ventilating 
slide,  in  order  that  any  moisture  left  in  the  tubes,  etc., 
may  escape. 

3.  When  the  temperature  of  the  oven  has  reached 
100°  C.,  close  the  ventilating  slide;  reset  the  alarm  to 
ring  at  175°  C. 

4.  Run  the  temperature  up  to  175°  C, 


MOIST   HEAT.  37 

5.  Extinguish  the  gas  at  once,  and  allow  the  appa- 
ratus to  cool. 

6.  When  the  temperature  of  the  interior,  as  recorded 
by  the  thermometer,  has  fallen  to  60°  C., — but  not 
before, — the  door  may  be  opened  and  the  sterile  articles 
removed  and  stored  away. 

N.  B. — Neglect  of  this  precautionary  cooling  of  the 
oven  to  60°  C.  will  result  in  numerous  cracked  and 
broken  tubes. 

On  removal  from  the  oven,  the  cotton- wool  plugs  will 
probably  be  slightly  brown  in  colour. 

Metal  instruments,  such  as  knives,  scissors,  and 
forceps,  may  be  sterilised  in  the  hot-air  oven  as  de- 
scribed above,  but  exposure  to  175°  C.  is  likely  to 
seriously  affect  the  temper  of  the  steel  and  certainly 
blunts  the  cutting  edges.  If,  however,  it  is  desired 
to  sterilise  surgical  instruments  by  hot  air,  they  should 
be  packed  in  a  metal  box,  or  boxes,  and  heated  to 
130°  C.  and  retained  at  that  temperature  for  about 
thirty  minutes. 

Moist  Heat.— Water  at  56°  C.— This  temperature,  if 
maintained  for  thirty  minutes,  is  sufficient  to  destroy 
the  vegetative  forms  of  bacteria,  but  has  practically 
no  effect  on  spores.  Its  use  is  limited  to  the  sterilisa- 
tion of  such  albuminous  "fluid"  media  as  would  co- 
agulate at  a  higher  temperature. 

METHOD.— 

1.  Fit  up  a  water-bath,  heated  by  a  Bunsen  flame 
which  is  controlled  by  a  thermo-regulator,  so  that  the 
temperature  of  the  water  remains  at  56°  C. 

2.  Immerse  the  tubes  or  flasks  containing  the  albu- 
minous fluid  in  the  water-bath  so  that  the  upper  level 
of  such  fluid  is  at  least  2  cm.  below  the  level  of  the 
water.     (The  temperature  of  the  bath  will  now  fall 
somewhat,  but  after  a  few  minutes  will  again  rise  to 
56°  C.) 


38  METHODS    OF    STERILISATION. 

3.  After  thirty  minutes'   exposure  to   56°  C.,   ex- 
tinguish the  gas,  remove  the  tubes  or  flasks  from  the 
bath,  and  subject  them  to  the  action  of  running  water 
so  that  their  contents  are  rapidly  cooled. 

4.  The  vegetative  forms  of  bacteria  present  in  the 
liquid  being  killed,  stand  it  for  twenty-four  hours  in 
a  cool,  dark  place;  at  the  end  of  that  time  some  at  least 
of  the  spores  will  have  germinated  and  assumed  the 
vegetative  form. 

5.  Destroy  these  new  vegetative  forms  by  a  similar 
exposure  to  56°  C.  on  the  second  day,  whilst  others, 
of  slower  germination,  may  be  caught  on  the  third  day, 
and  so  on. 

6.  In  order  to  ensure  thorough  sterilisation,  repeat 
the  process  on  each  of  six  successive  days. 

This  method  of  exposing  liquids  to  a  temperature 
of  56°  C.  in  a  water-bath  for  half  an  hour  on  each  of 
six  successive  days  is  termed  fractional  sterilisation. 

Water  at  100°  C.  destroys  the  vegetative  forms  of 
bacteria  almost  instantaneously,  and  spores  in  from 
five  to  fifteen  minutes.  This  method  of  sterilisation 
is  applicable  to  the  metal  instruments,  such  as  knives, 
forceps,  etc.,  used  in  animal  experiments,  syringes, 
rubber  corks,  rubber  and  glass  tubing,  and  other  small 
apparatus,  and  is  effected  in  what  is  usually  spoken  of 
as  the  "water  steriliser." 

This  is  a  rectangular  copper  box,  26  cm.  long,  18  cm, 
wide,  and  12  cm.  deep,  mounted  on  legs,  heated  from 
below  by  a  Bunsen  or  radial  gas  burner,  and  containing 
a  movable  copper  wire  tray,  2  cm.  smaller  in  every 
dimension  than  the  steriliser  itself,  and  which  is  pro- 
vided with  handles.  The  top  of  the  steriliser  is  hinged 
to  form  a  lid. 

METHOD. — 

i.  Place  the  instruments,  etc.,  to  be  sterilised  inside 
the  copper  basket,  and  replace  the  basket  in  the  ster- 
iliser. 


STREAMING    STEAM. 


39 


2.  Pour  a  sufficient  quantity  of  water  into  the  ster- 
iliser, shut  down  the  lid,  and  light  the  gas  below. 

3.  After  the  water  has  boiled  and  steam  has  been 
issuing  from  beneath  the  lid  for  ten  minutes,  extinguish 
the  gas,  open  the  lid,  and  lift  out  the  wire  basket  by  its 
handles;    the    contained    instruments,   etc.,    are    now 
sterile  and  ready  for  use. 

4.  After  use,   or  when  accidentally  contaminated, 
replace  the  instruments  in  the  basket  and  return  that 


Fig.  19. — Koch's  steriliser. 


Fig.  20. — Arnold's  steriliser. 


to  the  steriliser ;  completely  disinfect  by  a  further  boil- 
ing for  fifteen  minutes. 

5.  After  disinfection,  and  whilst  still  hot,  take  out 
the  instruments,  carefully  dry  them  at  once,  and  return 
them  to  their  store  cases. 

Streaming  steam — i.  e.,  steam  at  100°  C. — destroys 
the  vegetative  forms  of  bacteria  in  from  fifteen  to 
twenty  minutes,  and  the  sporing  forms  in  from  one 
to  two  hours.  This  method  is  chiefly  used  for  the 
sterilisation  of  the  various  nutrient  media  intended  for 


40  METHODS    OF   STERILISATION. 

the  cultivation  of  bacteria,  and  is  carried  out  in  a 
steam  kettle  of  special  construction,  known  as  Koch's 
steam  steriliser  (Fig.  19)  or  in  one  of  its  many  modifi- 
cations, the  most  efficient  of  which  is  Arnold's  (Fig.  20). 

The  steam  steriliser  in  its  simplest  form  consists  of 
a  tall  tinned-iron  or  copper  cylindrical  vessel,  divided 
into  two  unequal  parts  by  a  movable  perforated  metal 
diaphragm,  the  lower,  smaller  portion  serving  for  a 
water  reservoir,  and  the  upper  for  the  reception  of 
wire  baskets  containing  the  articles  needing  sterilisa- 
tion. The  vessel  is  closed  by  a  loose  conical  lid,  pro- 
vided with  handles,  and  perforated  at  its  apex  by  a 
tubulure;  it  is  mounted  on  a  tripod  stand  and  heated 
from  below  by  a  Bunsen  burner.  The  more  elaborate 
steriliser  is  cased  with  felt  or  asbestos  board,  and  pro- 
vided with  a  water  gauge,  also  a  tap  for  emptying  the 
water  compartment. 

To  USE  THE  STEAM  STERILISER. — 

1.  Fill  the  water  compartment  to  the  level  of  the 
perforated  diaphragm,  place  the  lid  in  position,  and 
light  the  Bunsen  burner. 

2.  After  the  water  has  boiled,  allow  sufficient  time 
to  elapse  for  steam  to  replace  the  air  in  the  sterilising 
compartment,   as  shown  by  the  steam  issuing  in  a 
steady,  continuous  stream  from  the  tubulure  in  the  lid. 

3.  Remove  the  lid,  quickly  lower  the  wire  basket 
containing  media  tubes,  etc.,  into  the  sterilising  com- 
partment until  it  rests  on  the  diaphragm,  and  replace 
the  lid. 

4.  After  an  interval  of  twenty  minutes  in  the  case 
of  fluid  media,  or  thirty  minutes  in  the  case  of  solid 
media,  take  off  the  lid  and  remove  the  basket  with  its 
contents.  ,  • 

5.  Now,  but  not  before,  extinguish  the  gas. 

NOTE. — After  removing  tubes,  flasks,  etc.,  from  the 
steam  steriliser,  they  should  be  at  once  separated 
freely  in  order  to  prevent  moisture  condensing  upon 


SUPERHEATED   STEAM.  41 

the  cotton-wool  plugs  and  soaking  through  into  the 
interior  of  the  tubes. 

This  treatment  will  destroy  any  vegetative  forms  of 
bacteria ;  during  the  hours  of  cooling  any  spores  present 
will  germinate,  and  the  young  organism  will  be  de- 
stroyed by  repeating  the  process  twenty-four  hours 
later;  a  third  sterilisation  after  a  similar  interval  makes 
assurance  doubly  sure. 

The  method  of  sterilising  by  exposure  to  streaming 
steam  at  100°  C.  for  twenty  minutes  on  each  of  three 
consecutive  days  is  termed  discontinuous  or  intermittent 
sterilisation. 

Continuous  sterilisation,  or  exposure  to  steam  at 
100°  C.  for  a  period  of  one  or  two  hours,  is  not  to  be 
recommended. 

Superheated  steam — i.  e.,  steam  under  pressure  in 
sealed  vessels  at  a  temperature  of  115°  C. — will  destroy 
both  the  vegetative  and  the  sporing  forms  of  bacteria 
within  fifteen  minutes;  if  the  pressure  is  increased, 
and  the  temperature  raised  to  120°  C.,  the  same  end 
is  attained  in  ten  minutes.  This  method  was  formerly 
employed  for  the  sterilisation  of  media,  but  when  it 
was  realised  that  hydrolytic  changes  occurred  in  media 
subjected  to  this  high  temperature,  which  rendered 
them  unfit  for  the  cultivation  of  the  more  delicate  micro- 
organisms, its  use  was  restricted  almost  entirely  to 
the  disinfection  of  such  contaminated  articles,  old  cul- 
tivations, etc.,  as  could  not  be  dealt  with  by  dry  heat 
or  the  actual  furnace.  Sterilisation  by  means  of  super- 
heated steam  is  carried  out  in  a  special  boiler — Cham- 
berland's  autoclave  (Fig.  21).  The  autoclave  consists 
of  a  stout  copper  cylinder,  provided  with  a  copper  or 
gun-metal  lid,  which  is  secured  in  place  by  means  of 
bolts  and  thumbscrews,  the  joint  between  the  cylinder 
and  its  lid  being  hermetically  sealed  by  the  interposi- 
tion of  a  rubber  washer.  The  cover  is  perforated  for 
a  branched  tube  carrying  a  vent  cock,  a  manometer,  and 


METHODS   OF   STERILISATION. 


a  safety  valve.  The  copper  boiler  is  mounted  in  the 
upper  half  of  a  cylindrical  sheet-iron  case — two  con- 
centric circular  rows  of  Bunsen  burners,  each  circle 
having  an  independent  gas-supply,  occupying  the 
lower  half.  In  the  interior  of  the  boiler  is  a  large  mov- 
able wire  basket,  mounted 
on  legs,  for  the  reception 
of  the  articles  to  be  ster- 
ilised. 

To    USE    THE    AUTO- 
CLAVE.— 

1.  Pack  the  articles  to 
be   sterilised   in  the  wire 
basket. 

2.  Run  water  into  the 
boiler  to  the  level  of  the 
bottom  of  the  basket ;  also 
fill    the    contained    flasks 
and  tubes  with  water. 

3.  See  that  the  rubber 
washer  is  in  position,  then 
replace     the     cover    and 
fasten  it  tightly  on  to  the 
autoclave  by  means  of  the 
thumbscrews. 

4.  Open  the  vent  cock  and  light  both  rings  of  burners. 

5.  When  steam  is  issuing  in  a  steady,  continuous 
stream  from  the  vent  tube,  shut  off  the  vent  cock  and 
extinguish  the  outer  ring  of  gas  burners. 

6.  Wait  until  the  index  of  the  manometer  records  a 
temperature  of  120°  C.,  then  regulate  the  gas  and  the 
spring  safety  valve  in  such  a  manner  that  this  tem- 
perature is   just   maintained,  and  leave   it   thus   for 
twenty  minutes. 

7.  Extinguish   the  gas   and   allow  the  manometer 
index  to  fall  to  zero. 

8.  Now  open  the  vent  cock  slowly,  and  allow  the 


Fig.  21. — Autoclave. 


FILTERS.  43 

internal  pressure  to  adjust  itself  to  that  of  the  atmos- 
phere. 

9.  Remove  the  cover  and  take  out  the  sterilised  con- 
tents. 

Filters.  —  (a)  Cotton-wool.  —  Practically  the  only 
method  in  use  in  the  laboratory  for  the  sterilisation 
of  air  or  of  a  gas  is  by  filtration  through  dry  cotton- 
wool or  glass-wool,  the  fibres  of  which  entangle  the 
micro-organisms  and  prevent  their  passage. 

Perhaps  the  best  example  of  such  a  filter  is  the  cotton- 
wool plug  which  closes  the  mouth  of  a  culture  tube. 
Not  only  does  ordinary  diffusion  take  place  through  it, 
but  if  a  tube  plugged  in  the  usual  manner  with  cotton- 
wool is  removed  from  the  hot  incubator,  the  tempera- 
ture of  the  contained  air  rapidly  falls  to  that  of  the 


Fig.  22. — Air  filter. 

laboratory,  and  a  partial  vacuum  is  formed;  air  passes 
into  the  tube,  through  the  cotton- wool  plug,  to  restore 
the  equilibrium,  and,  so  long  as  the  plug  remains  dry, 
in  a  germ-free  condition.  If,  however,  the  plug  be- 
comes moist,  either  by  absorption  from  the  atmosphere, 
or  from  liquids  coming  into  contact  with  it,  micro- 
organisms (especially  the  mould  fungi)  commence  to 
multiply,  and  the  long  thread  forms  rapidly  penetrate 
the  substance  of  the  plug,  gain  access  to  and  contami- 
nate the  interior  of  the  tube. 

If  it  is  desired  to  sterilise  gases  before  admission  to 
a  vessel  containing  a  pure  cultivation  of  a  micro- 
organism, as,  for  instance,  when  forcing  a  current  of 
oxygen  over  or  through  a  broth  cultivation  of  the 


44 


METHODS    OF    STERILISATION. 


diphtheria  bacillus,   this  can  be  readily  effected   as 
follows : 

1.  Take  a  length  of  glass  tubing  of,  say,   1.5  cm. 
diameter,  in  the  centre  of  which  a  bulb  has  been  blown, 
fill  the  bulb  with  dry  cotton- wool,  wrap  a  layer  of 
cotton- wool  around  each  end  of  the  tube,  and  secure 
in  position  with  a  turn  of  thread  or  string;  then  ster- 
ilise the  piece  of  apparatus  in  the  hot-air  oven. 

2.  Prepare  the  cultivation  in  a  Ruffer  or  Woodhead 
flask  (Fig.  23)  the  inlet  tube    of   which   has  a  layer 
of   cotton-wool    wrapped   round   it    and    secured  by 


Fig.  23.— Ruffer' s  flask. 

thread,  whilst  the  exit  tube  is  plugged  in  the  usual 
manner. 

3.  Sterilise  a  short  length  of  rubber  tubing  by  boil- 
ing.    Transfer  it  from  the  boiling  water  to  a  beaker 
of  absolute  alcohol. 

4.  Remove  the  rubber  tube  from  the  alcohol  by 
means  of  a  pair  of  forceps,  drain  it  thoroughly,  and 
pass  through  the  flame  of  a  Bunsen  burner  to  burn  off 
the  last  traces  of  alcohol. 

5.  Remove  the  cotton- wool  wraps  from  the  entry 
tube  of  the  flask  and  from  one  end  of  the  filter  tube 
and  rapidly  couple  them  up  by  means  of  the  sterile 
rubber  tubing. 


APPARATUS   REQUIRED. 


45 


6.  Connect  the  other  end  of  the  bulb  tube  with  the 
delivery  tube  from  the  gas  reservoir. 

The  gas  in  its  passage  through  the  dry  sterile  cotton- 
wool in  the  bulb  of  the  filter  tube  will  be  freed  from 
any  contained  micro-organisms  and  will  enter  the 
flask  in  a  sterile  condition. 

(b)  Porcelain. — The  sterilisation  of  liquids  by  filtra- 
tion is  effected  by  passing  them  through  a  cylindrical 
vessel,  closed  at  one  end  like  a  test-tube,  and  made 
either  of  porous  "biscuit"  porcelain,  hard-burnt  and 
unglazed  (Chamberland  system),  or  of  Kieselguhr,  a 
fine  diatomaceous  earth  (Berkfeld 
system),  and  termed  a  "bougie"  or 
"candle"  (Fig.  24). 

In  this  method  the  bacteria  are  re- 
tained in  the  pores  of  the  filter  while 
the  liquid  passes  through  in  a  germ- 
free  condition. 

It  is  obvious  that  to  be  effective 
the  pores  of  the  filter  must  be  ex- 
tremely minute,  and  therefore  the 
rate  of  filtration  will  usually  be  slow. 
To  overcome  this  defect,  aspiration  or 
pressure,  or  a  combination  of  the  two, 
may  be  employed  to  hasten  the  process. 

T-,         .     j  Fig.  24. — Filter 

Apparatus  Required. —  &  candle. 

1.  Separatory  funnel    containing   the 
unfiltered  fluid. 

2.  Sterile  filter  candle,  the  open  end  fitted  with  a  rubber 
stopper  perforated  to  receive  the  delivery  tube  of  the  sep- 
aratory  funnel,  and  its  neck  passed  through  a  large  rubber 
washer  (a)  which  fits  the  mouth  of  the  filter  flask. 

3.  Sterile  filter  flask  of  suitable  size,  for  the  reception  of 
the  filtered  fluid,  its  mouth  closed  by  a  cotton-wool  plug. 

4.  Water  injector  pump,  or  Geryk's  pump  (an  air  pump 
on  the  hydraulic  principle,  sealed  by  means  of  low  vapor- 
tension  oil,  Fig.  25). 

If  this  latter  is  employed,  a  Wulff's  bottle,  fitted  as  a 
wash-bottle  and  containing  sulphuric  acid,  must  be  inter- 
posed between  the  filter  flask  and  the  pump,  in  order  to 
prevent  moist  air  reaching  the  oil  in  the  pump. 


46 


METHODS    OF   STERILISATION. 


5.  Air  filter  (vide  page  43)  sterilised. 

6.  Pressure  tubing. 

7.  Screw  clamps  (Fig.  26). 


To  filter  flask 


Fig.  25. — Geryk  air  pump. 


METHOD. — 

i.  Couple  the  exhaust  pipe  of  the  suction  pump 
with  the  lateral  tube  of  the  filter  flask  (first  removing  the 

cotton-wool  plug  from  this 
latter),  by  means  of  pressure 
tubing,  interposing,  if  neces- 
sary, the  wash-bottle  of  acid. 
2.  Remove  the  cotton- wool 
plug   and  adjust  the  porce- 
Fig.  26.— Screw  clamps.         lain  candle  in  the  neck  of  the 

filter  flask. 

3.  Attach  the  nozzle  of  the  separatory  funnel  to  the 
filter  candle  by  means  of  the  perforated  rubber  stopper 
(Fig.  27). 


TO   FILTER.  47 

4.  Open  the  tap  of  the  funnel,  and  exhaust  the  air 
from  the  filter  flask  and  wash-bottle;  maintain  the 
vacuum  until  the  filtration  is  complete. 

5.  Adjust   a   screw  clamp  to   the   pressure   tubing 
attached   to   the   lateral   branch   of   the  filter   flask; 
screw  it   up   tightly,  and  disconnect  the  acid  wash- 
bottle. 

6.  Attach  the  air  filter  to  the  open  end  of  the  pressure 
tubing;  open  the  screw  clamp  gradually,  and  allow 


To  exhaust 
pump 


Fig.  27. — Apparatus  arranged  for  filtering — aspiration. 

filtered  air  to  enter  the  flask,  to  destroy  the  negative 
pressure. 

7.  Detach  the  rubber  tubing  from  the  lateral  branch 
of  the  flask,  flame  the  end  of  the  branch  in  the  Bunsen, 
and  plug  its  orifice  with  sterile  cotton-wool. 

8.  Remove  the  filter  candle  from  the  mouth  of  the 
flask,  flame  the  mouth,  and  plug  with  sterile  cotton- 
wool. 


48 


METHODS  OF  STERILISATION. 


9.  Disinfect  the  filter  candle  and  separatory  funnel 
by  boiling. 

If  it  is  found  necessary  to  employ  pressure  in  addi- 
tion to  or  in  place  of  suction,  insert  a  perforated  rubber 
stopper  into  the  mouth  of  the  separatory  funnel  and 
secure  in  position  with  copper  wire;  next  fit  a  piece  of 
glass  tubing  through  the  stopper,  and  connect  the 
external  orifice  with  an  air-pressure  pump  of  some 
kind  (an  ordinary  foot  pump  such  as  is  employed  for 
inflating  bicycle  tires  is  one  of  the  most  generally  use- 


Fig.  28. — Apparatus  arranged  for  filtering. 


ful,  for  this  purpose)  or  with  a  cylinder  of  compressed 
air  or  other  gas. 

Some  forms  of  filter  candle  are  made  with  the  open 
end  contracted  into  a  delivery  nozzle,  which  is  glazed. 
In  this  case  the  apparatus  is  fitted  up  in  a  slightly 
different  manner;  the  fluid  to  be  filtered  is  contained 
in  an  open  cylinder  into  which  the  candle  is  plunged, 
while  its  delivery  nozzle  is  connected  with  the  filter 
flask  by  means  of  a  piece  of  flexible  pressure  tubing 
(previously  sterilised  by  boiling),  as  in  figure  28. 


TO   STERIUSE.  49 

In  order  to  filter  a  large  bulk  of  fluid  very  rapidly  it 
is  necessary  to  use  a  higher  pressure  than  glass  would 
stand,  and  in  these  cases  the  metal  receptacle  designed 
by  Pakes,  to  hold  the  filter  candle  itself  as  well  as  the 
fluid  to  be  filtered,  should  be  employed.  (A  vacuum 
should  also  be  maintained  in  the  filter  flask,  by  means 
of  an  exhaust  pump,  during  the  entire  process.) 

This  piece  of  apparatus  consists  of  a  brass  cylinder, 
capacity  2500  c.c.,  with  two  shoulders;  and  an  opening 
in  the  neck  at  each  end,  provided  with  screw  threads. 

A  nut  carrying  a  pressure  gauge  fits  into  the  top 
screw;  and  into  the  bottom  is  fitted  a  brass  cylinder 
carrying  the  filter  candle  and  prolonged  downwards 
into  a  delivery  tube.  Leakage  is  prevented  by  means 
of  rubber  washers. 

Into  the  top  shoulder  a  tube  is  inserted,  bent  at 
right  angles  and  provided  with  a  tap.  All  the  brass- 
work  is  tinned  inside  (Fig.  29,  a).  In  use  the  reservoir 
is  generally  mounted  on  a  tripod  stand. 

To  Sterilise.— 

1 .  Insert  the  filter  candle  into  its  cylinder  and  screw 
this  loosely  on. 

2.  Wrap  a  layer  of  cotton- wool  around  the  delivery 
tube  and  fasten  in  position. 

3.  Remove  the  nut  carrying  the  pressure  gauge  and 
plug  the  neck  with  cotton- wool. 

4.  Heat  the  whole  apparatus  in  the  autoclave  at 
120°  C.  for  twenty  minutes. 

METHOD.— 

1.  Remove  the  apparatus  from  the  autoclave,  and. 
allow  it  to  cool. 

2.  Screw  home  the  box  carrying  the  bougie. 

3.  Set  the  apparatus  up  in  position,  with  its  delivery 
tube  (from  which  the  cotton-wool  wrapping  has  been 
removed)  passing  through  a  perforated  rubber  stopper 
in  the  neck  of  a  filter  flask. 

4.  Fill  the  fluid  to  be  filtered  into  the  cylinder  and 

4 


50  METHODS   OF   STERILISATION. 

screw  on  the  nut  carrying  the  pressure  gauge.  (This 
nut  should  be  immersed  in  boiling  water  for  a  few 
minutes  previous  to  screwing  on,  in  order  to  sterilise  it.) 

5.  Connect  the  horizontal  arm  of  the  entry  tube 
with  a  cylinder  of  compressed  oxygen  (or  carbon  di- 
oxide, Fig.  29,  b),  by  means  of  pressure  tubing. 

6.  Connect  the  lateral  arm  of  the  filter  flask  with 


LJL.J 


Fig.  29 — Fakes'  filtering  reservoir— pressure  and  aspiration. 

the  exhaust  pump  (Fig.  29,  c)  and  start  the  latter  work- 
ing. 

7.  Open  the  tap  of  the  gas  cylinder;  then  open  the 
tap  on  the  entry  tube  of  the  filter  cylinder  and  raise 
the  pressure  in  its  interior  until  the  desired  point 
is  recorded  on  the  manometer.  Maintain  this  pressure 
until  filtration  is  completed,  by  regulating  the  tap  on 
the  entry  tube. 


m.  THE  MICROSCOPE. 

THE  essentials  of  a  microscope  for  bacteriological 
work  may  be  briefly  summed  up  as  follows: 

The  instrument  of  the  monocular  type  must  be  of 


Fig.  30. — Microscope  complete. 
51 


52  THE    MICROSCOPE. 

good  workmanship  and  well  finished,  rigid,  firm,  and 
free  from  vibration,  not  only  when  upright,  but  also 
when  inclined  to  an  angle  or  in  the  horizontal  position. 
The  various  joints  and  movements  must  work  smoothly 
and  precisely,  equally  free  from  the  defects  of  "loss 
of  time"  and  "slipping."  All  screws,  etc.,  should  con- 
form to  the  Royal  Microscopical  Society's  standard. 
It  must  also  be  provided  with  good  lenses  and  a  suffi- 
ciently large  stage.  The  details  of  its  component  parts, 
to  which  attention  must  be  specially  directed,  are  as 
follows : 

1.  The  Base  or  Foot  (Fig.  30,  a). — Two  elementary 
forms — the  tripod  (Fig.  3 1 ,  a)  and  the  vertical  column 


Fig.  31. — Foot,  three  types. 

set  into  a  plate  (Fig.  31,  b) — serve  as  the  patterns  for 
countless  modifications  in  shape  and  size  of  this  portion 
of  the  stand.  The  chief  desiderata — stability  and  ease 
of  manipulation — are  attained  in  the  first  by  means  of 
the  "spread"  of  the  three  feet,  which  are  usually  shod 
with  cork;  in  the  second,  by  the  dead  weight  of  the 
foot-plate.  The  tripod  is  mechanically  the  more 
correct  form,  and  for  practical  use  is  much  to  be  pre- 
ferred. Its  chief  rival,  the  Jackson  foot  (Fig.  31,  c),  is 
based  upon  the  same  principle,  and  on  the  score  of 
appearance  has  much  to  recommend  it. 

2.  The  body  tube  (Fig.  30,  b)  maybe  either  that  known 
as  the  "long"  or  "English"  (length  250  mm.),  or  the 
"short"  or  "Continental"  (length  160  mm.).  Neither 


COARSE    ADJUSTMENT. 


53 


length  appears  to  possess  any  material  advantage  over 
the  other,  but  it  is  absolutely  necessary  to  secure  objec- 
tives which  have  been  manufactured  for  the  particular 
tube  length  chosen.  In  the  high-class  microscope  of 
the  present  day  the  body  tube  is  usually  shorter  than 
the  Continental,  but  is  provided  with  a  draw  tube 
which,  when  fully  extended,  gives  a  tube  length  greater 


Fig.  32. — Coarse  adjustment. 


Fig.  33. — Fine  adjustment. 


than  the  English,  thus  permitting  the  use  of  either 
form  of  objective. 

(Optical  tube  length  =  distance  from  the  back  lens  of 
the  objective  to  the  field  glass  of  the  ocular. 

Mechanical  tube  length  —  distance  from  the  end  of  the 
nosepiece  to  the  eyeglass  of  the  ocular.) 

3.  The  coarse  adjustment  (Fig.  30,  c)  should  be  a 
rack-and-pinion  movement,  steadiness  and  smoothness 
of  action  being  secured  by  means  of  deeply  bevelled 
edges  and  careful  countersinking  (Fig.  32). 


54 


THE   MICROSCOPE. 


4.  The  fine  adjustment  (Fig.  30,  d)  should  on  no  ac- 
count depend  upon  the  action  of  springs,  but  should 
be  of  the  lever  pattern,  preferably  the  Nelson  (Fig.  33). 
In  this  form  the  unequal  length  of  the  arms  of  the  lever 
secures  very  delicate  movement,  and,  moreover,  only 
a  small  portion  of  the  weight  of  the  body  tube  is 
transmitted  to  the  thread  of  the  vertical  screw  actuat- 
ing the  movement. 

5.  The  stage  (Fig.  30,  e)  should  be  square  in  shape 
and  large  in  area, — at  least  12  cm., — flat  and  rigid,  in 


Fig.  34. — Mechanical  stage. 

order  to  afford  a  safe  support  for  the  Petri  dish  used  for 
plate  cultivations;  and  should  be  supplied  with  spring 
clips  (removable  at  will)  to  secure  the  3  by  i  glass  slides. 
A  mechanical  stage  must  be  classed  as  a  necessity 
rather  than  a  luxury  so  far  as  the  bacteriologist  is  con- 
cerned, as  when  working  with  high  powers,  and  espe- 
cially when  examining  hanging-drop  specimens,  it  is 
almost  impossible  to  execute  sufficiently  delicate  move- 
ments with  the  fingers.  In  selecting  a  mechanical 
stage,  preference  should  be  given  to  one  which  forms 


SUBSTAGE   CONDENSER.  55 

an  integral  part  of  the  ordinary  stage  (Fig.  34)  rather 
than  one  which  needs  to  be  clamped  on  every  time  it 
is  required.  The  mechanical  stage  should  be  fitted 
with  three  (removable)  screw  studs,  so  that  if  necessary 
a  Vernier  finder  (Fig.  34,  D),  such  as  is  usually  fitted 
to  this  class  of  stage,  or  a  Maltwood  finder,  may  be 
used. 

6.  Diaphragm. — Separate  single  diaphragms  must 
be  avoided;  a  revolving  plate  pierced  with  different- 
sized  apertures  and  secured  below  the  stage  is  prefer- 
able, but  undoubtedly  the  best  form  is  the  "iris" 
diaphragm  (Fig.  35). 


ig-  35-  —  Diaphragm  iris. 


7.  The  substage  condenser  is  a  necessary  part  of  the 
optical  outfit.  Its  purpose  is  to  collect  the  rays  of 
light  reflected  by  the  mirror,  by  virtue  of  a  short  focus 
system  of  lenses,  into  a  cone  of  large  aperture  (redu- 
cible at  will  by  means  of  an  iris  diaphragm  mounted 
as  a  part  of  the  condenser),  which  can  be  accurately 
focussed  on  the  plane  of  the  object.  This  focussing 
must  be  performed  anew  for  each  object,  on  account 
of  the  variation  in  the  thickness  of  the  slides. 

The  form  in  most  general  use  is  that  known  as  the 
Abbe  (Fig.  36)  and  consists  of  a  plano-convex  lens 
mounted  above  a  biconvex  lens.  This  combination  is 
carried  in  a  screw-centering  collar  below  the  stage  of 


THE    MICROSCOPE. 


Fig-  36.— Optical  part  of  Abbe 
illuminator. 


the  microscope  (Fig.  30,  /),  and  must  be  accurately 
adjusted  so  that  its  optical  axis  coincides  with  that  of 

the  objective.  Vertical  move- 
ment of  the  entire  substage 
apparatus  effected  by  means 
of  a  rack  and  pinion  is  a  de- 
cided advantage,  and  some 
means  should  be  provided  for 
temporarily  removing  the 
condenser  from  the  optical 
axis  of  the  microscope. 

8.  Mirrors. — Below  the  substage    condenser  is  at- 
tached a  reversible   circular  frame  bearing  a  plane 
mirror  on  one  side  and  a  concave  mirror  on  the  other 
(Fig.  30,  g) .    The  plane  mirror  is  that  usually  employed, 
but  occasionally,  as  for  example  when  using  low  powers 
and  with  the  condenser  racked  down  and  thrown  out 
of  the  optical  axis,  the  concave  mirror  is  used. 

9.  Oculars,    or    Eyepieces. — Those    known    as    the 
Huyghenian  oculars  (Fig.  37)  will  be  sufficient  for  all 
ordinary   work    without  resorting 

to  the  more  expensive  ' '  compensa- 
tion" oculars.  Two  or  three,  mag- 
nifying the  "real"  image  (formed 
by  the  objective)  four,  six,  or  eight 
times  respectively,  form  a  useful 
equipment. 

10.  Objectives.  —  Three    objec- 
tives are  necessary:  one  for  low- 
power  work — e.  g.,  i  inch,  f  inch, 
or    \   inch;     one    for    high-power 
work — e.  g.,  ^  inch  oil  immersion 
lens;  and  an  intermediate  lens — 
e.  g.,\  inch  or  J  inch  (dry).     These 
lenses  must  be  carefully  selected, 

especial  attention  being  paid  to  the  following  points: 
(a)    Correction    of   Spherical   Aberration. — Spherical 


Fig.  37. — Huyghenian 
eyepiece. 


NUMERICAL   APERTURE.  57 

aberration  gives  rise  to  a  distorted  image,  due  to  the 
central  and  peripheral  rays  focussing  at  different  points. 
(6)  Correction  of  Chromatic  Aberration. — Chromatic 
aberration  gives  rise  to  a  coloured  fringe  around  the 
edge  of  the  field,  which  is  due  to  the  fact  that  the 
different-coloured  rays  of  the  spectrum  possess  vary- 
ing refrangibilities  and  that  a  simple  lens  acts  towards 
them  as  a  prism. 

(c)  Flatness  of  Field. — The  visual  field  should  be 
large  and,  above  all,  flat;  in  other  words,  objects  at  the 
periphery  of  the  field  should  be  as  distinctly  "  in  focus" 
as  those  in  the  centre.     Failing  this,  the  lens  should 
possess  a  large  central  "flat"  area  and  the  entire  per- 
ipheral ring  should  come  into  focus  at  the  same  moment 
and  with  the  least  possible  movement  of  the  fine  ad- 
justment. 

(d)  Good  Definition. — Actual  magnification  is,  within 
limits,  of  course,  of  less  value  than  clear  definition  and 
high  resolving  power,  for  it  is  upon  these  properties 
we  depend  for  our  knowledge  of  the  detailed  structure 
of  the  objects  examined. 

(e)  Numerical   Aperture    (N .    A.). — The   numerical 
aperture  may  be  defined,  in  general  terms,  as  the  ratio 
of  the  effective  diameter  of  the  back  lens  of  the  objective 
to  its  equivalent  focal  length.     The  determination  of 
this  point  is  a  process  requiring  considerable  technical 
skill    and    mathematical    ability,    and    is    completely 
beyond  the  powers  of  the  average  microscopist.1 

Although  with  the  increase  in  power  it  is  increasingly 
difficult  to  combine  all  these  corrections  in  one  objec- 
tive, they  are  brought  to  a  high  pitch  of  excellence 
in  the  present-day  "  achromatic"  objectives,  and  so 

1  Its  importance  will  be  realised,  however,  when  it  is  stated  in  the  words  of 
Professor  Abbe :  "  The  numerical  aperture  of  a  lens  determines  all  its 
essential  qualities  ;  the  brightness  of  the  image  increases  with  a  given  magni- 
fication and  other  things  being  equal,  as  the  square  of  the  aperture  ;  the  resolv- 
ing and  defining  powers  are  directly  related  to  it,  the  focal  depth  of  differentia- 
tion of  depths  varies  inversely  as  the  aperture,  and  so  forth." 


58  THE  MICROSCOPE. 

remove  the  necessity  for  the  use  of  the  higher  priced 
and  less  durable  apochromatic  lenses. 

In  selecting  objectives  the  best  "test"  objects  to 
employ  are: 

1.  A  thin  (one  cell  layer),  even  "  blood  film,"  stained 
with  eosin  and  counterstained  with  methylene-blue. 

2.  A  thin  cover-slip  preparation  of  a  young  culti- 
vation of  the  B.  diphtheria  (showing  segmentation) 
stained  with  methylene-blue. 

Accessories. — Nosepiece. — The  first  and  most  useful 
accessory  is  a  nosepiece  to  carry  two  of  the  objectives 
(Fig.  38),  or,  better  still,  all  three  (Fig.  39).  This 
nosepiece,  preferably  constructed  of  aluminium,  is 
of  the  covered-in  type,  consisting  of  a  curved  plate 


Fig.  38. — Double  nosepiece.  Fig.  39. — Triple  nosepiece. 

attached  to  the  lower  end  of  the  body  tube — a  circular 
aperture  being  cut  to  correspond  to  the  lumen  of  that 
tube.  To  the  under  surface  of  this  plate  is  pivoted  a 
similarly  curved  plate,  fitted  with  three  tubulures, 
each  of  which  carries  an  objective.  By  rotating  the 
lower  plate  each  of  the  objectives  can  be  brought  suc- 
cessively in  to  the  optical  axis  of  the  microscope. 

Warm  Stage  (Fig.  40). — This  is  a  flat  metal  case 
through  the  interior  of  which  water  of  any  required 
temperature  can  be  circulated.  It  is  made  to  clamp 
on  to  the  stage  of  the  microscope,  and  is  perforated 
with  a  large  hole  coinciding  with  the  optical  axis  of  the 
microscope;  and  by  raising  the  temperature  of  hang- 
ing-drop preparations,  etc.,  placed  upon  it,  above  that 
of  the  surrounding  atmosphere,  is  extremely  useful 


MICROMETER. 


59 


for  observations  on  spore  germination,  hanging- drop 
cultivations,  etc. 

Eye  Shade  (Fig.  41). — This  piece  of  apparatus  con- 
sists of  a  pear-shaped  piece  of  blackened  metal,  hinged 


Fig.  40. — Warm  stage. 

to  a  collar  which  rotates  on  the  upper  part  of  the 
body  tube  of  the  microscope.  It  can  be  used  to  shut 
out  the  image  of  surrounding  objects  from  the  un- 
occupied eye,  and  when  carrying  out  prolonged  obser- 
vations will  be  found  of  real  service. 


Fig.  41. — Eye  screen. 

Micrometer. — Some  form  of  micrometer  for  the  pur- 
pose of  measuring  bacteria  and  other  objects  is  also 
essential.  Details  of  those  in  general  use  will  be  found 
in  the  following  pages. 


60  THE  MICROSCOPE. 

METHODS  OF  MICROMETRY. 

The  unit  of  length  as  applied  to  the  measurement 
of  microscopical  objects  is  the  one-thousandth  part 
of  a  millimetre  (o.ooi  mm.),  denominated  a  micron 
(sometimes,  and  erroneously,  referred  to  as  a  micro- 
millimetre),  and  indicated  in  writing  by  the  Greek 
letter  //.  Of  the  many  methods  in  use  for  the  measure- 
ment of  bacteria,  three  only  will  be  here  described, 
viz.: 

By  means  of  the  stage  micrometer. 


Fig.  42. — Camera  lucida,  Abbe  pattern. 

By  means  of  the  ocular  or  eyepiece  micrometer. 

By  means  of  the  filar  micrometer  (Ramsden's  mi- 
crometer eyepiece). 

(a)  By  means  of  the  stage  micrometer. 

The  stage  micrometer  is  a  3  by  i  inch  glass  slip  having 
engraved  on  it  a  scale  divided  to  hundredths  of  a  milli- 
metre (o.oi  mm.),  every  tenth  line  being  made  longer 
than  the  intervening  ones,  to  facilitate  counting.  A 
cover-glass  is  cemented  over  the  scale  to  protect  it 
from  injury. 


EYEPIECE    MICROMETER. 


6l 


1 .  Attach  a  camera  lucida  (of  the  Wollaston,  Beale, 
or  Abbe  pattern)  to  the  eyepiece  of  the  microscope. 

2.  Adjust  the  micrometer  on  the  stage  of  the  micro- 
scope and  accurately  focus  the  divisions. 

3.  Project  the  scale  of  the  stage  micrometer  on  to 
a  piece  of  paper  and  with  pen  or  pencil  sketch  in  the 
magnified  image,  each  division  of  which  corresponds 
to   10  ii.     Mark  on  the  paper  the  optical  combination 
(ocular  objective  and  tube  length)  employed  to  pro- 
duce this  particular  magnification. 

4.  Repeat  this  procedure  for  each  of  the  possible 
combinations  of  oculars  and  objectives  fitted  to  the 
microscope  supplied,  and  carefully  preserve  the  scales 
thus  obtained. 


Fig.  43. — Eyepiece  micrometer, 
ordinary. 


Fig.  44. — Eyepiece  micrometer,  net. 


To  measure  an  object  by  this  method  simply  project 
the  image  on  to  the  scale  corresponding  to  the  par- 
ticular optical  combination  in  use  at  the  moment. 
Read  off  the  number  of  divisions  it  occupies  and  ex- 
press them  as  micra. 

In  place  of  preserving  a  scale  for  each  optical  com- 
bination, the  object  to  be  measured  and  the  micrometer 
scale  may  be  projected  and  sketched,  in  turn,  on  the 
same  piece  of  paper. 

(6)   By  means  of  the  eyepiece  micrometer. 

The  eyepiece  micrometer  is  a  circular  glass  disc  having 
engraved  on  it  a  scale  divided  to  tenths  of  a  millimetre 
(o.i  mm.)  (Fig.  43),  or  the  entire  surface  ruled  in  o.i 


62  THE   MICROSCOPE. 

mm.  squares  (the  net  micrometer)  (Fig.  44).  It  can  be 
fitted  inside  the  mount  of  any  ocular  just  above  the 
aperture  of  the  diaphragm  and  must  be  adjusted 
exactly  in  the  focus  of  the  eyeglass. 

Some  makers  mount  the  glass  disc  together  with  a 
circular  cover-glass  in  such  a  way  that  when  placed 
in  position  in  any  Huyghenian  eyepiece  of  their  own 
manufacture,  the  scale  is  exactly  in  focus. 

The  value  of  one  division  of  the  micrometer  scale 
must  first  be  ascertained  for  each  optical  combination 
by  the  aid  of  the  stage  micrometer,  thus : 

1.  Insert  the  eyepiece  micrometer  inside  the  ocular 
and  adjust  the  stage  micrometer  on  the  stage  of  the 
microscope. 

2.  Focus  the  scale  of  the  stage  micrometer  accurately; 
the  lines  will  appear  to  be  immediately  below  those  of 
the  eyepiece  micrometer.     Make  the  lines  on  the  two 
micrometers  parallel  by  rotating  the  ocular. 

3.  Make  two  of  the  lines  on  the  ocular  micrometer 
coincide  with  those  bounding  one  division  of  the  stage 
micrometer;  this  is  effected  by  increasing  or  diminish- 
ing the  tube  length;  and  note  the  number  of  included 
divisions. 

4.  Calculate  the  value  of  each  division  of  the  eye- 
piece  micrometer  in  terms  of  /*,  by  means  of  the  fol- 
lowing formula: 

x  =  10  y. 
Where  x  =  the  number  of  included  divisions  of  the 

eyepiece  micrometer. 

y  =  the  number  of  included  divisions  of  the 
stage  micrometer. 

5.  Note  the  optical  combination  employed  in  this 
experiment  and  record  it  with  the  calculated  microm- 
eter value. 

Repeat  this  process  for  each  of  the  other  combina- 
tions. Carefully  record  the  results. 

To  measure  an  object  by  this  method  read  off  the 


FILAR   MICROMETER.  63 

number  of  divisions  of  the  eyepiece  micrometer  it 
occupies  and  express  the  result  in  micra  by  a  refer- 
ence to  the  standard  value  for  the  particular  optical 
combination  employed. 

Zeiss  prepares  a  compensating  eyepiece  micrometer 
for  use  with  his  apochromatic  objectives,  the  divisions 
of  which  are  so  computed  that  (with  a  tube  length  of 
1 60  mm.)  the  value  of  each  is  equivalent  to  as  many 
micra  as  there  are  millimetres  in  the  focal  length  of  the 
objective  employed. 

(c)   By  means  of  the  filar  micrometer. 

The  filar  or  cobweb  micrometer  (Ramsden's  microm- 


Fig.  45. — Ramsden's  micrometer. 


Fig.  46. — Ramsden's 
micrometer  field. 


eter  eyepiece  (Fig.  45)  consists  of  an  ocular  having  a 
fine  "  fixed"  wire  stretching  horizontally  across  the 
field  (Fig.  46),  a  vertical  reference  wire — fixed — ad- 
justed at  right  angles  to  the  first;  and  a  fine  wire,  paral- 
lel to  the  reference  wire,  which  can  be  moved  across 
the  field  by  the  action  of  a  micrometer  screw;  the 
trap  head  is  divided  into  one  hundred  parts,  which 
successively  pass  a  fixed  index  as  the  head  is  turned. 
In  the  field  is  also  fixed  a  comb  with  the  intervals  be- 
tween its  teeth  corresponding  to  one  complete  revolu- 
tion of  this  screw-head. 


64  THE   MICROSCOPE. 

As  in  the  previous  method,  the  value  of  each  division 
of  the  micrometer  scale  (i.  e.,  the  comb)  must  first  be 
determined  for  each  optical  combination.  This  is 
effected  as  follows: 

1 .  Place  the  filar  micrometer  and  the  stage  microm- 
eter in  their  respective  positions. 

2.  Rotate  the  screw  of  the  filar  micrometer  until  the 
movable  wire  coincides  with  the  fixed  one,  and  the 
index  marks  zero  on  the  screw-head. 

3.  Focus  the  scale  of  each  micrometer  accurately, 
and  make  the  lines  on  them  parallel. 

4.  Rotate  the  head  of  the  micrometer  screw  until 
the  movable  line  has  traversed  one  division  of  the 
stage  micrometer.     Note  the  number  of  complete  revo- 
lutions (by  means  of  the  recording  comb)  and  the  frac- 
tions of  a  revolution  (by  means  of  scale  on  the  head 
of  the  micrometer  screw),  which  are  required  to  meas- 
ure the  o.o i  mm. 

5.  Make  several  such  estimations  and  average  the 
results. 

6.  Note  the  optical  combination  employed  in  this 
experiment  and  record  it  carefully,  together  with  the 
micrometer  value  in  terms  of  //. 

7.  Repeat  this   process   for   each   of   the   different 
optical  combinations  and  record  the  results. 

To  measure  an  object  by  this  method,  simply  note 
the  number  of  revolutions  and  fractions  of  a  revolu- 
tion of  the  screw-head  required  to  traverse  such  object 
from  edge  to  edge,  and  express  the  result  as  micra 
by  reference  to  the  recorded  values  for  that  particular 
optical  combination. 


IV.   MICROSCOPICAL   EXAMINATION  OF 
BACTERIA  AND  OTHER  MICRO-FUNGL 

APPARATUS  AND  REAGENTS  USED  IN  ORDINARY 
MICROSCOPICAL  EXAMINATION. 

EACH  student  is  provided  with  a  set  consisting  of 
the  following  pieces  of  apparatus  and  reagents. 

1.  India-rubber  "change-mat"   upon  which  cover- 
glasses  may  be  rested  during  the  process  of  staining. 

2.  Squares  of  blotting  paper  about  10  cm.,  for  dry- 
ing cover-slips  and  slides. 

(The  filter  paper  known  as  "German  lined" — a 
highly  absorbent,  closely  woven  paper,  having  an  even 
surface  and  no  loose  "  fluff"  to  adhere  to  the  specimens 
— is  the  most  useful  for  this  purpose.) 

3.  Glass  jar  filled  with  2  per  cent,  lysol  solution  for 
the   reception   of   infected   cover-glasses    and   useless 
slides. 

4.  Bunsen  burner  provided  with  by-pass. 

5.  Porcelain  trough  holding  five  or  six  hanging-drop 
slides  (Fig.  47). 

A  hanging-drop  slide  is  prepared  by  cementing  a 
circular  cell  of  tin,  13  to  15  mm.  diameter,  and  i  to  2 
mm.  in  height,  to  the  centre  of  a  3  by  i  slip  by  means 
of  Canada  balsam.  It  is  often  extremely  convenient 
to  have  two  of  these  cells  cemented  close  together  on 
one  slide. 

Another  form  of  hanging-drop  slide  is  made  in  which 
a  circular  or  oval  cell  is  ground  out  of  the  centre  of  a, 
3  by  i  slip.  These  are  more  expensive,  less  convenient 
to  work  with,  and  are  more  easily  contaminated  by 
drops  of  material  under  examination,  and  should  be 
carefully  avoided. 

5  65 


66 


MICROSCOPICAL    EXAMINATION. 


6.  Three  aluminium  rods  (Fig.  48),  each  about  25 
cm.  long  and  carrying  a  piece  of  0.015  gauge  platino- 
iridium  wire  7.5  cm.  in  length.  The  end  of  one  of  the 
wires  is  bent  round  to  form  an  oval  loop,  of  about  i 


Fig.  47. — Hanging-drop  slides :    a,  Double  cell  seen  from  above ;  b,  single 
cell  seen  from  the  side. 

mm.  in  its  short  diameter,  and  is  termed  a  loop  or  an 
ose;  the  terminal  3  or  4  mm.  of  another  wire  is  flat- 
tened out  by  hammering  it  on  a  smooth  iron  surface  to 
form  a  "spatula";  the  third  is  left  untouched  or  is 
pointed  by  the  aid  of  a  file.  These  instruments  are 


3C 


Fig.  48. — Ends  of  rods. 

used  for  inoculating  culture  tubes  and  preparing  speci- 
mens for  microscopical  examination. 

The  method  of  mounting  these  wires  may  be  de- 
scribed as  follows: 

Take  a  piece  of  aluminium  wire  25  cm.  long  and 


APPARATUS  AND  REAGENTS.  67 

about  0.25  cm.  in  diameter,  and  drill  a  fine  hole  com- 
pletely through  the  wire  about  a  centimetre  from  one 
end.  Sink  a  straight  narrow  channel  along  one  side 
of  the  wire,  in  its  long  axis,  from  the  hole  to  the  nearest 
end,  shallow  at  first,  but  gradually  becoming  deeper. 

On  the  opposite  side  of  the  wire  make  a  short  cut, 
2  mm.  in  length,  leading  from  the  hole  in  the  same 
direction. 

Now  pass  one  end  of  the  platinum  wire  through  the 
hole,  turn  up  about  2  mm.  at  right  angles  and  press 
the  short  piece  into  the  short  cut.  Turn  the  long  end 
of  the  wire  sharply,  also  at  right  angles,  and  sink  it 
into  the  long  channel  so  that  it  emerges  from  about 


Fig.  49. — Platinum  rod  in  aluminium  handle — method  of  mounting. 

the  centre  of  the  cut  end  of  the  aluminium  wire  (Fig. 
49).  A  few  sharp  taps  with  a  small  hammer  will 
now  close  in  the  side  of  the  two  channels  over  the  wire 
and  hold  it  securely. 

The  platinum  wire  may  be  fused  into  the  end  of  a 
piece  of  glass  rod,  but  such  a  handle  is  vastly  inferior 
to  aluminium  and  is  not  to  be  recommended. 

7.  Two  pairs  of  sharp-pointed  spring  forceps    (10 
cm.  long),  one  of  which  must  be  kept  perfectly  clean 
and  reserved  for  handling  clean  cover-slips,  the  other 
being  for  use  during  staining  operations. 

8.  A  box  of  clean  3  by  i  glass  slips. 

9.  A  glass  capsule  with  tightly  fitting  (ground  on) 


68 


MICROSCOPICAL    EXAMINATION. 


glass    lid,    containing    clean    cover-slips    in    absolute 
alcohol. 

10.  One  of  Faber's  "  grease  pencils"  (yellow,  red,  or 
blue)  for  writing  on  glass. 

11.  A  wooden   rack    fitted   with   ten    drop- bottles 
(Fig.  50)  each  60  c.c.  capacity,  containing 

Aniline  water. 

Gentian  violet,  saturated  alcoholic  solution. 

Gram's  iodine. 

Absolute  alcohol. 


Fig.  50. — Drop-bottle. 


Fig.  51. — Canada  balsam  pot. 


i  per  cent,  aqueous  solution, 
saturated  alcoholic ,  solution. 


Eosin,  yellowish, 

Methylene-blue, 

Fuchsin,  basic, 

Carbolic  acid,  5  per  cent,  aqueous  solution. 

Acetic  acid,  i  per  cent,  solution. 

Sulphuric  acid,  25  per  cent,  solution. 
And  two  pots  with  air-tight  glass  caps  (Fig.  51),  filled 
respectively  with  Canada  balsam  dissolved  in  xylol,  and 
sterile  vaseline,  and  each  provided  with  a  piece  of  glass 
rod. 


METHODS    OF    EXAMINATION.  69 

METHODS  OF  EXAMINATION. 
Bacteria,   etc.,  are  examined  microscopically,  both 

1.  In  the  living  state,  unstained,  or  stained. 

2.  After  having  been  fixed,  killed,  and  stained  by 
suitable  methods. 

The  preparation  of  a  specimen  from  a  tube  cultiva- 
tion for  examination  by  these  methods  may  be  de- 
scribed as  follows: 

1.  Living,  Unstained. — (a)  "Fresh"  Preparation. — • 
i.  Clean  and  dry  a  3  by  i  glass  slip  and  place  it  on 
one  of  the  squares  of  filter  paper.  Deposit  a  drop  of 
water  (preferably  distilled)  or  a  drop  of  i  per  cent, 
solution  of  caustic  potash,  on  the  centre  of  the  slip, 
by  means  of  the  platinum  loop. 


Fig.  52. — Holding  tubes  for  removing  cultivation,  as  seen  from  the  front. 

ft  2.  Take  the  tube  cultivation  in  the  left  hand 

and  ignite  the  cotton-wool  plug  by  holding  it 
to  the  flame  of  the  Bunsen  burner.  Extinguish 
the  flame  by  blowing  on  the  plug,  whilst  rotating 
the  tube  on  its  long  axis,  its  mouth  directed 
vertically  upwards,  between  the  thumb  and  fin- 
gers. (This  operation  is  termed  "flaming  the 
plug,"  and  is  intended  to  destroy  any  micro- 
organisms that  may  have  become  entangled  in 
^  the  loose  fibres  of  the  cotton- wool,  and  which, 
if  not  thus  destroyed,  might  fall  into  the  tube 


o  5 


70  MICROSCOPICAL   EXAMINATION. 

when  the  plug  is  removed  and  so  accidentally 
contaminate   the  cultivation.) 

3.  Hold  the  tube  at  or  near  its  centre  between 
the  ends  of  the  thumb  and  first  two  fingers  of 
the  left  hand,  and  allow  the  sealed  end  to  rest 
upon  the  back  of  the  hand  between  the  thumb 
and  forefinger,  the  plug  pointing  to  the  right. 
Keep  the  tube  as  nearly  in  the  horizontal  posi- 
tion as  is  consistent  with  safety,  to  diminish 
the  risk  of  the  accidental  entry  of  organisms 

g.  52). 

§  4.  Take  the  handle  of  the  loop  between  the 
thumb  and  forefinger  of  the  right  hand,  holding 
the  instrument  in  a  position  similar  to  that 
occupied  by  a  pen  or  a  paint-brush,  and  sterilise 
o  the  platinum  portion  by  holding  it  in  the  flame 
55  of  a  Bunsen  burner  until  it  is  red  hot.  Sterilise 
the  adjacent  portion  of  the  aluminium  handle 
by  passing  it  rapidly  twice  or  thrice  through 
the  flame.  After  sterilising  it,  the  loop  must  not 
be  allowed  to  leave  the  hand  or  to  touch  against 
anything  but  the  material  it  is  intended  to 
examine,  until  it  is  finished  with  and  has  been 
again  sterilised. 

5.  Grasp  the  cotton- wool  plug  of  the  test- 
tube  between  the  little  finger  and  the  palm  of 
the  right  hand  (whilst  still  holding  the  loop  as 
directed  in  step  4),  and  remove  it  from  the 
mouth  of  the  tube  by  a  "screwing"  motion  of 
the  right  hand. 

6.  Introduce  the  platinum  loop  into  the  tube 
and  hold  it  in  this  position  until  satisfied  that 
it  is  quite  cool.     (The  cooling  may  be  hastened 
by  touching  the  loop  on  one  of  the  drops  of 
moisture  which  are  usually  to  be  found  con- 
densed on  the  interior  of  the  glass  tube,  or  by 
dipping  it  into  the  condensation  water  at  the 


o 
g 

z 

w 

OH 

O 


K 
O 

w 


J 

u 


METHODS    OF    EXAMINATION.  71 

bottom;  at  the  same  time  care  must  be  taken 
in  the  case  of  cultures  of  solid  media  to  avoid 
touching  either  the  medium  or  the  growth.) 

7.  Remove  a  small  portion  of  the  growth  by 
taking  up  a  drop  of  liquid,  in  the  case  of  a  fluid 
culture,  in  the  loop  or  by  touching  it  on  the 
surface  of  the  growth  when  the  culture  is  on 
solid  medium ;  and  withdraw  the  loop  from  the 
tube  without  again  touching  the  medium  or 
the  glass  sides  of  the  tube. 

8.  Replace  the  cotton- wool  plug  in  the  mouth 
of  the  tube. 

9.  Mix   the   contents  of   the  loop  thoroughly  with 
the  drop  of  water  on  the  3  by  i  slide. 

10.  Again  sterilise  the  loop  as  directed  in  step  4, 
and  replace  it  in  its  stand. 

11.  Replace  the  tube  cultivation  in  its  rack  or  jar. 

12.  Remove  a  cover-slip  from  the  glass  capsule  by 
means  of  the  cover-slip  forceps,  rest  it  for  a  moment 
on  its  edge,  on  a  piece  of  filter  paper  to  remove  the 
excess  of  alcohol,  then  pass  it  through  the  flame  of  the 
Bunsen  burner.     This  burns  off  the  remainder  of  the 
alcohol,  and  the  cover-slip  so  "flamed"  is  now  clean, 
dry,  and  sterile. 

13.  I/ower  the  cover-slip,  still  held  in  the  forceps, 
on  to  the  surface  of  the  drop  of  fluid  on  the  3  by  i 
slip,  carefully  and  gently,  to  avoid  the  inclusion  of  air 
bubbles. 

14.  Examine  microscopically   (vide  infra). 
During  the  microscopical  examination,  stains  and 

other  reagents  may  be  run  in  under  the  cover-slips  by 
the  simple  method  of  placing  a  drop  of  the  reagent 
in  contact  with  one  edge  of  the  cover-glass  and  apply- 
ing the  torn  edge  of  a  piece  of  blotting  paper  to  the 
opposite  side.  The  reagent  may  then  be  observed 
to  flow  across  the  field  and  come  into  contact  with 
such  of  the  micro-organisms  as  lie  in  its  path. 


72  MICROSCOPICAL   EXAMINATION. 

(b)  Hanging-drop  Preparation. — 

1.  Smear  a  layer  of  sterile  vaseline  on  the  upper 
surface  of  the  ring  cell  of  a  hanging-drop  slide  by  means 
of  the  glass  rod  provided  with  the  vaseline  bottle,  and 
place  the  slide  on  a  piece  of  filter  paper. 

2.  "Flame"  a  cover- slip  and  place  it  on  the  filter 
paper  by  the  side  of  the  hanging-drop  slide. 

3.  Place  a  drop  of  water  on  the  centre  of  the  cover- 
slip  by  means  of  the  platinum  loop. 

4.  Obtain  a  small  quantity  of  the  material  it  is  de- 
sired to  examine,  in  the  manner  detailed  above  (steps 
2  to  ii  must  be  followed  in    their  entirety  and  with 
the  strictest  exactitude  whenever  tube  contents  are 
being  handled),  and  mix  it  with  the  drop  of  water  on 
the  cover-slip. 

5.  Raise  the  cover-slip  in  the  points  of  the  forceps 
and  rapidly  invert  it  on  to  the  ring  cell  of  the  hanging- 
drop  slide,  so  that  the  drop  of  fluid  occupies  the  centre 
of  the  ring.     (Carefully  avoid  contact  between  the 
drop  of  fluid  and  either  the  ring  cell  or  the  layer  of 
vaseline.    Should  this  happen,  the  now  infected  hanging- 
drop  slide  and  its  cover-slip  must  be  dropped  into  the 
pot  of  lysol  and  a  new  preparation  made.) 

6.  Press  the  cover-slip  firmly  down  into  the  vaseline 
on  to  the  top  of  the  ring  cell.     (This  spreads  out  the 
vaseline  into  a  thin  layer,  and  besides  ensuring  the 
adhesion  of  the  cover-slip,  seals  the  cells  and  so  retards 
evaporation.) 

7.  Examine  microscopically  (vide  infra). 
Microscopical  Examination  of  the  Unstained  Speci= 

mens.— 

1 .  Place  the  body  tube  of  the  microscope  in  the  ver- 
tical position. 

2.  Arrange  the  hanging-drop   slide  on   the  micro- 
scope stage  so  that  the  drop  of  fluid  is  in  the  optical 
axis  of  the  instrument,  and  secure  it  in  the  position 
by  means  of  the  spring  clips. 


EXAMINATION    OF    UNSTAINED    SPECIMENS.          73 

3.  Use  the  J-inch  objective,  rack  down  the  body 
tube  until  the  front  lens  of  the  objective  is  almost  in 
contact  with  the  cover-slip — that  is,  well  within  its 
focal  distance.     This  is  best  done  whilst  bending  down 
the  head  to  one  side  of  the  microscope,  so  that  the 
eyes  are  on  a  level  with  the  stage. 

4.  Apply  the  eye  to  the  ocular  and  adjust  the  plane 
mirror  to  the  position  which  secures  the  best  illumina- 
tion. 

5.  Rack  the  condenser  down  slightly  and  cut  down 
the  aperture  of  the  iris  diaphragm  so  that  the  light, 
although  even,  is  dim. 

6.  Rack  up  the  body  tube  by  means  of  the  coarse 
adjustment  until  the  bacteria  come  into  view;  then 
focus  exactly  by  means  of  the  fine  adjustment. 

Some  difficulty  is  often  experienced  at  first  in  finding 
the  hanging  drop,  and  if  the  first  attempt  is  unsuccess- 
ful, the  student  must  not  on  any  account,  whilst  still 
applying  his  eye  to  the  ocular,  rack  the  body  tube 
down  (for  by  so  doing  there  is  every  likelihood  of  the 
front  lens  of  the  objective  being  forced  through  the 
cover-glass,  and  not  only  spoiling  the  specimen,  but 
also  contaminating  the  objective) ;  but,  on  the  con- 
trary, withdraw  his  eye,  rack  the  tube  up,  and  com- 
mence again  from  step  2. 

The  examination  of  a  "fresh"  specimen  or  a  "hang- 
ing-drop" preparation  is  directed  to  the  determination 
of  the  following  data: 

1.  The  nature  of  the  bacteria  present — e.  g.,  cocci, 
bacilli,  etc. 

2.  The  purity  of  the  cultivation;  this  can  only  be 
determined  when  gross  morphological  differences  exist 
between  the  organisms  present. 

3.  The  presence  or  absence  of  spores,  which  show 
their    typical    refrangibility    exceedingly    well    when 
examined  by  this  method. 

4.  The  presence  or  absence  of  mobility.     In  a  hang- 


74  MICROSCOPICAL   EXAMINATION. 

ing-drop  specimen  some  form  of  movement  can  prac- 
tically always  be  observed,  and  its  character  must  be 
carefully  determined  by  noting  the  relative  positions 
of  adjacent  micro-organisms. 

(a)  Brownian  or  molecular  movement.  Minute  par- 
ticles of  solid  matter  (including  bacteria),  when  sus- 
pended in  a  fluid,  will  always  show  a  vibratory  move- 
ment affecting  the  entire  field,  but  never  altering  the 
relative  positions  of  the  bacteria.  (Cocci  exhibit  this 
movement,  but  with  the  exception  of  the  Micrococcus 
agilis,  the  cocci  are  non-motile.) 

(6)  Streaming  movement.  This  is  due  to  currents 
set  up  in  the  hanging  drop  as  a  result  of  jarring  of  the 
specimen  or  of  evaporation,  and  although  the  relative 
position  of  the  bacteria  may  vary,  still  the  flowing 
movement  of  large  numbers  of  organisms  in  some  one 
direction  will  usually  be  sufficient  to  demonstrate  the 
nature  of  this  motion. 

(c)  Locomotive  movement,  or  true  motility,  is  deter- 
mined by  observing  some  one  particular  bacillus  chang- 
ing its  position  in  the  field  independently  of,  and  in  a 
direction  contrary  to,  other  organisms  present. 

When  the  examination  is  completed  and  the  specimen 
finished  with,  the  "  fresh  specimen" — i.  e.,  the  slide  with 
the  cover-slip  attached — must  be  dropped  into  the 
lysol  pot.  In  the  hanging-drop  specimen,  however, 
the  cover-slip  only  is  infected,  and  this  may  be  raised 
from  the  ring  cell  by  means  of  forceps  and  dropped 
into  the  disinfectant. 

Permanent  Staining  of  the  Hanging-drop  Specimen. 
— Occasionally  it  is  necessary  to  fix  and  stain  a  hanging- 
drop  preparation.  This  may  be  done  as  follows: 

1.  Remove  the  cover- slip  from  the  cell  by  the  aid  of 
the  forceps. 

2.  If  the  drop  is  small,  fix  it  by  dropping  it  face 
downwards,  whilst  still  wet,  on  to  the  surface  of  some 
Gulland's  solution  or  corrosive  sublimate  solution  (vide 


KILLED,    STAINED.  75 

page  76)  in  a  watch-glass.  If  the  drop  is  large,  place 
it  face  upwards  on  the  rubber  mat,  cover  it  with  an 
inverted  watch-glass,  and  allow  it  to  dry.  Then  fix 
it  in  the  alcohol  and  ether  solution  (vide  infra) . 

3.  Dip  the  cover-glass  into  a  beaker  containing  hot 
water  in  order  to  remove  some  of  the  vaseline  adher- 
ing to  it. 

4.  Wash   successively  in  alcohol,  xylol,  ether,  and 
alcohol,  to  remove  the  last  traces  of  grease. 

5.  Wash  in  water. 

6.  Stain,  wash,  dry,  and  mount  as  for  an  ordinary 
cover-slip  film  preparation  (vide  page  78). 

2.  Killed,  Stained. — In  this  method  three  distinct 
processes  are  necessary: 

1.  "  Preparing"  and  "  fixing"  the  film. 

2.  Staining. 

3.  Mounting. 

i  a.  Preparing  the  Film. — 

1 .  Proceed  as  in  making  a  hanging-drop  preparation, 
steps  2  to  4. 

2.  Spread  the   drop   of  emulsion  evenly  over  the 
cover-slip  in  the  form  of  a  square  film  to  within  i  mm. 
of  each  edge  of  the  cover-slip. 

3.  Allow  it  to  dry  completely  in  the  air. 

i b.  Fixing. — Fix  by  passing  the  cover-slip,  held  in 
the  fingers,  three  times  through  the  flame  of  a  Bunsen 
burner.  In  preparing  films  for  staining,  it  is  sometimes 
necessary  (as  in  the  case  of  those  intended  for  micro- 
metric  observations)  to  fix  by  exposure  to  a  uniform 
temperature  of  115°  C.,  for  twenty  minutes.  This  is 
best  done  in  a  carefully  regulated  hot-air  oven.  Fixa- 
tion may  also  be  effected  by  immersing  in  some  fixative 
fluid,  such  as  one  of  the  following: 

1.  Absolute  alcohol. 

A  ,      ,  i     -L  i    f  equal  parts,  for  five  to  thirty 

2.  Absolute  alcohol,  .  J 

<      minutes  (e.  g.,  for  blood  or 

I     milk) . 


76  MICROSCOPICAL   EXAMINATION. 

3.  Osmic  acid,  i  per  cent,  aqueous  solution,  for  .thirty 
seconds. 

4.  Corrosive  sublimate,  saturated  aqueous  solution, 
for  five  minutes. 

5.  Corrosive   sublimate    (I^ang),    for  five    minutes. 
This  solution  is  prepared  by  dissolving: 

Sodium  chloride 0.75  gramme 

Hydrarg.  perchlor 12.00  grammes 

Acetic  acid 5.00        " 

In  distilled  water loo.oo  c.c. 

Filter. 

6.  Gulland's  solution,  for  five  minutes.     This  solu- 
tion is  prepared  by  mixing : 

Absolute  alcohol 25.0  c.c. 

Ether 25.0    " 

Corrosive  sublimate,  20  per  cent,  alcoholic  solution     .    0.4    " 

Either  of  these  methods  of  fixation  coagulates  the 
albuminous  material  and  ensures  perfect  adhesion  of 
the  film  to  the  cover-slip. 

Wash  the  cover-slip  thoroughly  in  running  water  and 
proceed  with  the  staining. 

If  the  film  has  been  prepared  from  broth,  liquefied 
gelatine,  or  pus  or  other  morbid  exudations,  saturate 
the  film  after  fixation  with  acetic  acid  2  per  cent,  and 
allow  it  to  act  for  two  minutes. 

Wash  with  alcohol,  then  let  the  alcohol  remain  on 
the  cover-slip  for  two  minutes.  (This  will  "clear" 
the  groundwork  and  give  a  much  sharper  and  cleaner 
film  than  would  otherwise  be  obtained.) 

If  the  film  has  been  prepared  from  blood  or  blood- 
stained fluid,  treat  with  acetic  acid  2  per  cent,  for  two 
minutes  after  fixation.  Wash  with  water,  dry,  and 
proceed  with  the  staining.  (This  will  remove  the 
haemoglobin  and  facilitate  examination.) 

2.  Staining. — 

i.  Rest  the  cover-slip,  film  side  uppermost,  on  the 
rubber  mat. 


STAINING.  77 

2.  By  means  of  a  drop-bottle,  cover  the  film  side  of 
the  cover-slip  with  the  selected  stain,  allow  it  to  act 
for  a  few  minutes,  then  wash  off  the  excess  in  running 
water. 

The  penetrating  power  of  stains  is  increased  by  (a) 
physical  means — e.  g.,  heating  the  stain;  (b)  chemical 
means — e.  g.,  by  the  addition  of  carbolic  acid,  5  per 
cent,  aqueous  solution;  caustic  alkalies,  2  per  cent, 
aqueous  solution;  water  saturated  with  aniline  oil; 
borax,  0.5  per  cent,  aqueous  solution. 

The  most  commonly  used  dyes  for  cover-slip  film 
preparations  are  the  aniline  dyes. 

(A)  Basic: 

(a)  Methylene-blue. 

(b)  Gentian  violet. 

(c)  Fuchsin. 

These  dyes  are  kept  in  saturated  alcoholic  (90  per 
cent.)  solutions  so  that  decomposition  may  be  re- 
tarded. 

Two  or  three  drops  of  alcoholic  solution  of  these 
dyes  to,  say,  4  c.c.  water,  usually  makes  a  sufficiently 
strong  staining  fluid  for  cover-slip  film  preparations. 

Carbolic  methylene-blue  (C.M.B.)  and  carbol  f uchsin 
(C.F.)  are  prepared  by  covering  the  cover-slip  with  5 
per  cent,  solution  of  carbolic  acid  and  adding  a  few 
drops  of  the  saturated  alcoholic  solution  of  methylene- 
blue  or  f  uchsin  respectively  to  it.  For  aniline  gentian 
violet  (A.G.V.)  the  stain  is  added  to  a  saturated  solu- 
tion of  aniline  oil  in  water. 

(d)  Thionin  blue. 

(e)  Bismarck  brown. 

(B)  Acid: 

(a)  Eosin,  aqueous  yellowish. 

(b)  Safranine. 

These  dyes  are  kept  in  i  per  cent,  aqueous  solution 
to  which  is  added  5  per  cent,  of  alcohol,  as  a  preserva- 
tive. They  are  generally  used  in  this  form. 


78  MICROSCOPICAL  EXAMINATION. 

A  few  nuclear  stains  (carmine,  haemal oxylin)  are 
occasionally  used  more  especially  in  " section"  work. 

2a.  Decolonisation. — After  overstaining,  films  may 
be  decolourised  by  washing  for  a  longer  or  shorter  time 
in  one  of  the  following  reagents  arranged  in  ascending 
order  of  power 

1.  Water. 

2.  Chloroform. 

3.  Acetic  acid,  i  per  cent. 

4.  Alcohol. 

5.  Alcohol  absolute,  | 
Acetic  acid,  i  per  cent.,  / 

Hydrochloric,  i  per  cent,  aqueous 

solution. 
Hydrochloric,   i    per   cent,  alco- 


6.  Mineral  acids 


holic  (90  per  cent.)  solution. 


Sulphuric,  25  per  cent,  aqueous 

solution. 

Nitric,  33  per  cent,  aqueous  solu- 
tion. 

2b.  Counter  staining. — Use    colours   which  will  con- 
trast with  the  first  stain;  e.  g., 

^^         '  (  for  films  stained  by  methylene-blue  or 
Eosin,       V      ~        ,          •      ,  • 
_^         .      (      Gram  s  method. 
Fuchsm,  ) 

Methylene-blue,  ")  r      £1 

~  .  t         >  for  films  stained  by  fuchsm. 

Gentian  violet,    j 

3.  Mounting.— 

1.  Wash  the  film  carefully  in  running  water. 

2.  Blot  off  the  superfluous   water  with  the  filter 
paper,  or  dry  more  completely  between  two  folds  of 
blotting  paper. 

3.  Complete  the  drying  in  the  air,  or  by  holding  the 
cover-slip  in  the  fingers  at  a  safe  distance  above  the 
flame  of  the  Bunsen  burner. 

4.  Place  a  drop  of  xylol  balsam  on  the  centre  of  a 
clean  3  by  i  glass  slide  and  invert  the  cover-slip  over 


IMPRESSION    FILMS.  79 

the  balsam,  and  lower  it  carefully  to  avoid  the  inclusion 
of  air  bubbles. 

NOTE. — Xylol  is  used  in  preference  to  chloroform  to 
dissolve  Canada  balsam,  as  it  does  not  decolourise 
the  specimen. 

Impression  films  (Klatschprdparat)  are  prepared 
from  isolated  colonies  of  bacteria  in  order  that  their 
characteristic  formation  may  be  examined  by  higher 
powers  than  can  be  brought  to  bear  on  the  living  culti- 
vation. They  are  prepared  from  plate  cultivations 
(vide  page  181),  in  the  following  manner. 

1.  Remove  a  clean  cover-slip  from  the  alcohol  pot 
with  sterile  forceps  and  burn  off  the  spirit. 

2.  Open  the  plate  and  rest  one  edge  of  the  cover- 
slip  on  the  surface  of  the  medium  a  little  to  one  side 
of  the  selected  colony.     I/ower  it  cautiously  over  the 
colony  until  horizontal.     Avoid  any  lateral  movement 
or  the  inclusion  of  bubbles  of  air. 

3.  Make  gentle  vertical  pressure  on  the  centre  of  the 
cover-slip  with  the  points  of  the  forceps  to  ensure 
perfect  contact  with  the  colony. 

4.  Steady  one  edge  of  the  cover-slip  with  the  forceps 
and  pass  the  point  of  a  mounted  needle  just  under 
the  opposite  edge  and  raise  the  cover-slip  carefully; 
the  colony  will  be  adherent  to  it.     When  nearly  verti- 
cal, grasp  the  cover-slip  with  the  forceps  and  remove 
it  from  the  plate.     Re-cover  the  plate. 

5.  Place  the  cover-slip,  film  uppermost,  on  the  rubber 
mat,  and  cover  it  with  an  inverted  watch-glass  until 
dry. 

6.  Fix  by  immersing  in  one  of  the  fixing  fluids  pre- 
viously mentioned  (vide  page  75). 

7.  Clear  with  acetic  acid  and  alcohol. 

8.  Stain  and  mount  as  an  ordinary  cover-slip  film 
preparation,  being  careful  to  perform  all  washing  op- 
erations with  extreme  gentleness. 


80  MICROSCOPICAL   EXAMINATION. 

Microscopical  Examination  of  the  Stained  Specimen. 

— (The  body  tube  of  the  microscope  may  be  vertical 
or  inclined  to  an  angle.) 

1 .  Secure  the  slide  on  the  stage  of  the  microscope  by 
means  of  the  spring  clips. 

2.  Place  a  drop  of  cedarwood  oil  on  the  centre  of  the 
cover-slip. 

3.  Use  the  y^-inch  oil  immersion  lens  of  the  micro- 
scope.    Rack  down  the  body  tube  till  the  front  lens 
of  the  objective  is  in  contact  with  the  oil  and  nearly 
touching  the  cover-slip. 

4.  Rack  up  the  condenser  until  it  is  in  contact  with 
the  under  surface  of  the  slide. 

5.  Apply  the  eye  to  the  ocular  and  arrange  the  plane 
mirror  so  as  to  obtain  the  greatest  possible  amount  of 
light. 

6.  Rack  up  the  body  tube  until  the  stained  film 
comes  into  view. 

7.  Focus  the  condenser  accurately  on  the  film. 

8.  Focus  the  film  accurately  by  means  of  the  fine 
adjustment. 


V.  STAINING  METHODS. 

BACTERIA  STAINS. 

IN  the  following  pages  are  collected  the  various 
"stock"  stains  in  everyday  use  in  the  bacteriological 
laboratory,  together  with  a  selection  of  the  most  con- 
venient and  generally  useful  staining  methods  for 
demonstrating  particular  structures  or  differentiating 
groups  of  bacteria.  The  stains  employed  should  either 
be  those  prepared  by  Griibler,  of  Leipzig,  or  Merck,  of 
Darmstadt.  The  methods  printed  in  ordinary  type 
are  those  which  a  long  experience  has  shown  to  be  the 
most  reliable,  and  to  give  the  best  results 

Methylene=blue. — 

1.  Saturated  Aqueous  Solution. 
Weigh  out 

Methylene-blue      1.5  grammes 

Place  in  a  stoppered  bottle  having  a  capacity  of 
from  150  to  200  c.c.  and  add 

Distilled  water 100.0  c.c. 

Allow  the  water  to  remain  in  contact  with  the  dye 
for  two  weeks,  shaking  the  contents  of  the  bottle  vig- 
ourously  for  a  few  moments  every  day.  Filter. 

2.  Saturated  Alcoholic  Solution. 
Weigh  out 

Methylene-blue  .    .        1.5  grammes 

Place  in  a  stoppered  bottle  of  150  c.c.  capacity  and 
add 

Alcohol,  90  per  cent 100.0  c.c. 

Allow  the  alcohol  to  remain  in  contact  with  the  dye 
for  two  hours,  shaking  vigourously  every  few  minutes. 
Filter. 

6  81 


82  STAINING   METHODS. 

3.  Carbolic  Methylene-blue  (Kiihne). 
Weigh  out 

Methylene-blue 1.5  grammes 

Carbolic  acid  .    .    . 5.0        " 

and  dissolve  in 

Distilled  water 100.0  c.c. 

and  add 

Absolute  alcohol 10.0   " 

Filter. 

4.  Alkaline  Methylene-blue  (Loffler). 
Measure  out  and  mix 

Methylene-blue,  saturated  alcoholic  solution  .    .     30.0  c.c. 
Caustic  potash,  o. I  per  cent,  aqueous  solution   .  100.0    " 

Filter. 

Fuchsin  (Basic). — 

5.  Saturated  Aqueous  Solution. 
Weigh  out 

Basic  fuchsin 1.5  grammes 

and  proceed  as  in  preparing  the  corresponding  solution 
of  methylene-blue  (q.  v.). 

6.  Saturated  Alcoholic  Solution. 
Weigh  out 

Basic  fuchsin 3.5  grammes 

and  proceed  as  in  preparing  the  corresponding  solu- 
tion of  methylene-blue. 

7.  Carbolic  Fuchsin  (Ziehl). 
Weigh  out 

Basic  fuchsin i.o  gramme 

Carbolic  acid 5.0  grammes 

dissolve  in 

Distilled  water 100.0  c.c. 

and  add 

Absolute  alcohol 10.0   " 

Filter. 


CONTRAST   STAINS.  83 

Gentian  Violet. — 

8.  Saturated  Aqueous  Solution. 
Weigh  out 

Gentian  violet ; 2.25  grammes 

and  proceed  as  in  preparing  the  corresponding  solution 
of  methylene-blue. 

9.  Saturated  Alcoholic  Solution. 
Weigh  out 

Gentian  violet 5.0  grammes 

and  proceed  as  in  preparing  the  corresponding  solu- 
tion of  methylene-blue. 

10.  Carbolic  Gentian  Violet  (Nicolle). 
Measure  out  and  mix 

Gentian  violet,  saturated  alcoholic  solution      .    .     10.0  c.c. 
Carbolic  acid,  I  per  cent,  aqueous  solution      .    .  100.0    " 

Filter. 

Thionine  Blue  (or  Lauth's  Violet).— 

11.  Carbolic  Thionine  Blue  (Nicolle). 
Weigh  out 

Thionine  blue      . i.o  gramme 

Carbolic  acid 2.5  grammes 

and  dissolve  in 

Distilled  water 100.0  c.c. 

Filter. 

Before  use  dilute  with  equal  quantity  of  distilled 
water  and  again  filter. 

CONTRAST  STAINS. 

Eosin. — There  are  several  commercial  varieties  of 
eosin,  which,  from  the  bacteriological  point  of  view, 
possess  very  different  values.  Griibler  lists  four  varie- 
ties, of  which  two  only  are  useful  for  bacteriological 
work: 

Eosin,  aqueous  yellowish. 

Eosin,  aqueous  bluish. 


84  STAINING   METHODS. 

12.  Eosin  Aqueous  Solution   (Yellowish  or   Bluish 
Shade),    i    per   cent. 

Weigh  out 

Eosin,  aqueous i.o  gramme 

dissolve  in 

Distilled  water loo.o  c.c. 

and  add 

Absolute  alcohol 5.0    " 

Filter. 

13.  Eosin  Alcoholic  Solution,  0.5  per  cent. 
Weigh  out 

Eosin,  alcoholic 0.5  gramme 

and  dissolve  in 

Alcohol  (70  percent.) 100.0  c.c. 

Filter. 

Vesuvin  (or  Bismarck  Brown). — 

14.  Saturated  A queous  Solution. 
Weigh  out 

Vesuvin 0.5  gramme 

and  dissolve  in 

Distilled  water . 100.0  c.c. 

Filter. 
Safranine. — 

Weigh  out 

Safranine 0.5  gramme 

and  dissolve  in 

Distilled  water 100.0  c.c. 

Filter. 

TISSUE  STAINS. 
Lithium  Carmine  (Orth). — 
Weigh  out 

Carmine 2.5  grammes 

and  dissolve  in 

Lithium  carbonate,  cold  saturated  solution  .    .    .  loo.o  c.c. 

Filter. 
Picrocarmine. — 

Weigh  out 

Picrocarmine 2.0  grammes 

and  dissolve  in 

Distilled  water  loo.o  c.c. 


TISSUE   STAINS.  85 

Hsematoxylin. — 

1 .  Weigh  out 

Hsematoxylin 2.0  grammes 

and  dissolve  in 

Absolute  alcohol loo.o  c.c. 

2.  Weigh  out 

Ammonium  alum 2.0  grammes 

and  dissolve  in 

Distilled  water 100.0  c.c. 

3.  Mix  i  and  2,  allow  the  mixture  to  stand  forty- 
eight  hours,  then  filter. 

4.  Add 

Glycerine 85.0  c.c. 

Acetic  acid,  glacial 10.0   " 

5.  Allow  the  stain  to  stand  for  one  month;  then 
filter  ready  for  use. 

Aniline  Gentian  Violet  (For  Weigert's  Fibrin  Stain). — 
Weigh  out 

Gentian  violet i.o  gramme 

and   dissolve   in 

Absolute  alcohol      15.0  c.c. 

Distilled  water 80.0   " 

then  add 

Aniline  oil 3.0   " 

Shake  well  and  filter  before  use. 
Alum  Carmine  (Meyer). — 
Weigh  out 

Alum 2.5  grammes 

Carmine I.o  gramme 

and  place  in  a  glass  beaker. 
Measure  out  in  a  measuring  cylinder, 

Distilled  water 100.0  c.c. 

Place  the  beaker  on  a  sand-bath,  add  the  water  in 
successive  small  quantities,  and  keep  the  mixture'boil- 
ing  for  twenty  minutes.  Measure  the  solution  and 
make  up  to  100  c.c.  by  the  addition  of  distilled  water. 
Filter. 


86  STAINING   METHODS. 

METHODS  OF  DEMONSTRATING  STRUCTURE  OF 
BACTERIA. 

To  Demonstrate  Capsules. 
1.  MacConkey. — 

Stain. — 
Weigh  out 

Dahlia 0.5  gramme 

Methyl  green  (oo  crystals) 1.5  grammes 

rub  up  in  a  mortar  with 

Distilled  water 100.0  c.c. 

Add 

Fuchsin,  saturated  alcoholic  solution 10.0    " 

and  make  up  to  200  c.c.  by  the  addition  of 

Distilled  water 90.0  c.c. 

Filter. 

Allow  the  stain  to  stand  for  two  weeks  before  use; 
keep  in  a  dark  place  or  in  an  amber  glass  bottle.  Owing 
to  the  unstable  character  of  the  methyl  green,  this 
stain  deteriorates  after  about  six  months. 

METHOD. — , 

1 .  Prepare  and  fix  film  in  the  usual  manner. 

2.  Flood  the  cover-slip  with  the  stain  and  allow  it 
to  act  for  five  to  ten  minutes. 

3.  Wash  very  thoroughly  in  water;  if  necessary, 
direct  a  powerful  stream  of  water  on  the  film  from  a 
wash-bottle. 

4.  Dry  and  mount. 

2.  Welch's  Method.— 

1.  Prepare  and  fix  film  in  the  usual  manner. 

2.  Flood  the  slide  with  acetic  acid  2  per  cent.;  allow  the 
acid  to  remain  in  contact  with  the  film  for  two  minutes. 
This  swells  up  and  fixes  the  capsule  and  enables  it  to  take 
the  §tain. 

3.  Blow  off  the  acetic  acid  by  the  aid  of  a  pipette. 

4.  Immerse  in  aniline  gentian  violet,  five  to  thirty  seconds. 

5.  Wash  in  water. 

6.  Dry  and  mount. 


TO    DEMONSTRATE    FLAGEU/A.  87 

3.  Ribbert's  Method.— 

Stain. — 

Measure  out  and  mix 

Acetic  acid,  glacial      12.5  c.c. 

Alcohol,  absolute 50.0    " 

Distilled  water loo.o    " 

Warm  to  36°  C.  (e.  g.y  in  the  "hot"  incubator)  and  satu- 
rate with  dahlia.     Filter. 
METHOD.— 

1.  Prepare  and  fix  films  in  the  usual  manner. 

2.  Cover  the  film  with  the  stain  and  allow  it  to  act  for  one 
or  two  seconds  only. 

3.  Wash  thoroughly  in  water. 

4.  Dry  and  mount. 

To  Demonstrate  Flagella. 

1.  Muir's  Modified  Pitfield.— This  is  the  best  method 
and  gives  the  most  reliable  results,  for  not  only  is  the 
percentage  of  successful  preparations  higher  than  with 
any  other,  but  the  bacilli  and  flagella  retain  their  rela- 
tive proportions. 

(a)  Mordant. — 

Tannic  acid,  10  per  cent,  aqueous  solution  .    .    .  10  c.c. 

Corrosive  sublimate,  saturated  aqueous  solution  .  5    " 

Alum,  saturated  aqueous  solution      5    '« 

Carbolic  fuchsin  (Ziehl) 5    " 

Mix  thoroughly. 

A  precipitate  forms  which  must  be  allowed  to  settle 
for  a  few  hours. 

Decant  off  the  clear  fluid  into  tubes  and  centrif  ugalise 
thoroughly. 

This  solution  keeps  for  about  a  couple  of  weeks, 
but  must  be  re-centrifugalised  each  time,  before  use. 

(b)  Stain.— 

Alum,  saturated  aqueous  solution 25  c.c. 

Gentian  violet,  saturated  alcoholic  solution  ...       5    " 

Filter. 

This  stain  must  be  freshly  prepared. 
METHOD. — The    cultivations    employed    should    be 
smear  agar  cultures,  twelve  to  eighteen  hours  old  if 


88  STAINING   METHODS. 

incubated  at  37°  C.,  twenty-four  to  thirty  hours  if 
incubated  at  22°  C. 

1.  Remove  a  very  small  quantity  of  the  growth  by 
means  of  the  platinum  spatula. 

2.  Emulsify  it  with  a  few  cubic  centimetres  of  dis- 
tilled water  in  a  watch-glass,  by  gently  moving  the 
spatula  to  and  fro  in  the  water.     Do  not  rub  up  the 
growth  on  the  side  of  the  watch-glass. 

3.  Spread  a  thin  film  of  the  emulsion  on  a  newly 
flamed  cover-slip,  using  no  force,  but  rather  leading 
the  drop  over  the  cover-slip  with  the  platinum  loop. 

4.  Allow  the  film  to  dry  in  the  air,  properly  protected 
from  falling  dust. 

5.  Fix  by  passing  thrice  through  the  Bunsen  flame, 
holding  the  cover-slip  whilst  doing  so  by  one  corner 
between  the  finger  and  thumb. 

6.  Pour  on  the  film  as  much  of  the  mordant  as  the 
cover-glass  will  hold.     Grasp  the  cover-slip  with  the 
forceps  and  hold  it,  high  above  the  flame,  until  steam 
rises.     Allow  the  steaming  mordant  to  remain  in  con- 
tact with  the  film  two  minutes. 

7.  Wash  well  in  water  and  dry  carefully. 

8.  Pour  on  the  film  as  much  of  the  stain  as  the  cover- 
glass  will  hold.     Steam  over  the  flame  as  before  for 
two  minutes. 

9.  Wash  well  in  water. 

10.  Dry  and  mount. 

2.  •«  Pitfield  "  Original  Method.^- 

(a)  Mordant. — 

Tannic  acid I  gramme 

Water 10  c.c. 

(6)  Stain.— 

Saturated  aqueous  solution  of  alum   ......     10  c.c. 

Saturated  alcoholic  solution  of  gentian  violet  .    .       I    " 
Distilled  water 5    " 

Mix  equal  parts  of  a  and  b  before  using. 

i.  Prepare  and  fix  the  film  in  the  manner  described  above. 


VAN  ERMENGEM'S  METHOD.  89 

2.  Boil  the  mixture  and  immerse  the  cover-slip  in  it, 
whilst  still  hot,  for  one  minute. 

3.  Wash  in  water. 

4.  Examine  in  water;  if  satisfactory,  dry  and  mount  in 
Canada  balsam. 

3.  MacCrorrie's  Method. — 
Mordant-Stain. — 
Measure  out  and  mix 

Night  blue,  saturated  alcoholic  solution    ....     IO  c.c. 
Potash  alum,  saturated  aqueous  solution  ....     IO    " 
Tannin,  10  per  cent,  aqueous  solution 10    " 

NOTE. — The  addition  of  gallic  acid,  o.i  to  0.2  gramme, 
may  improve  the  solution,  but  is  not  necessary. 
METHOD. — 

1.  Prepare  and  fix  the  films  as  above. 

2.  Pour  some  of  the  mordant-stain  on  the  film  and  warm 
gently,  high  above  the  flame,  for  two  minutes  (or  place  in 
the  "hot"  incubator  for  a  like  period). 

3.  Wash  thoroughly  in  water. 

4.  Dry  and  mount. 

4.  Van  Ermengem's  Method. — This  method,  being 
merely  a  precipitation  of  a  silver  salt  on  the  micro-organ- 
isms and  not  a  true  stain,  is  unsatisfactory;  the  relative  pro- 
portions of  bacteria  and  flagella  are  almost  invariably  de- 
stroyed. ' 

(a)  Fixing  Fluid. — 

Osmic  acid,  2  per  cent,  aqueous  solution  .    ,    .    .     10  c.c. 
Tannic  acid,  20  per  cent,  aqueous  solution  ...     20    " 
Acetic  acid,  glacial i    " 

The  fixing  fluid  should  be  prepared  some  days  before  use 
and  filtered  as  required.  In  colour  it  should  be  distinctly 
violet. 

(6)  Sensitising  Solution. — 

Silver  nitrate,  o.  5  per  cent,  aqueous  solution. 

This  solution  must  be  kept  in  a  dark  blue  glass  bottle  or 
in  a  dark  cupboard. 

Filter  immediately  before  use. 
(c)  Reducing  Solution. — 
Weigh  out 

Gallic  acid 5  grammes 

Tannic  acid      3       " 

Potassium  acetate,  fused 10       " 

and  dissolve  in 

Distilled  water 350  c.c. 

Filter. 


90  STAINING   METHODS. 

This  solution  will  keep  active  for  several  days,  but  fresh 
solution  must  be  used  for  each  preparation. 
METHOD.  — 

1.  Prepare  emulsion,  make  and  fix  films  therefrom  as  de- 
scribed in  the  preceding  method,  steps  i  to  4. 

2.  Pour  on  the  film  as  much  of  the  fixing  solution  as  the 
cover-glass  will  hold,  heat  carefully  over  the  flame  till  steam 
rises,   and  allow  the  steaming  fixing  fluid  to  act  for  five 
minutes. 

3.  Wash  well  in  water. 

4.  Wash  in  absolute  alcohol. 

5.  Wash  in  distilled  water. 

6.  Pour  some  of  the  sensitising  solution  on  the  film  and 
allow  it  to  act  for  from  thirty  seconds  to  one  minute;  blot 
off  the  excess  of  fluid  with  filter  paper. 

7.  Without  washing,  transfer  the  film  to  a  watch-glass 
containing  the  reducing  solution  and  allow  it  to  remain 
therein  for  from  thirty  seconds  to  one  minute;  blot  off  the 
excess  of  fluid  with  filter  paper. 

8.  Without  washing,  again  treat  the  film  with  the  sensi- 
tising solution,  this  time  until  the  film  commences  to  turn 
black. 

9.  Wash  in  distilled  water. 

10.  Dry  and  mount. 

5.  Loffler's  Method.— 
(a)  Mordant.  — 

Tannic  acid,  20  per  cent,  aqueous  solution  .    .    .     10  c.c. 

Ferrous  sulphate,  saturated  aqueous  solution   .    .       5    " 

Hsematoxylin  solution  (prepared  by  boiling  I 
gramme  logwood  with  8  c.c.  distilled  water, 
filtering  and  replacing  the  loss  from  evapora- 
tion) .................  3  " 

Carbolic  acid,  I  per  cent,  aqueous  solution  ...      4    " 

This  solution  must  be  freshly  prepared. 
Alternative  Mordant.  — 

Tannic  acid,  20  per  cent,  aqueous  solution  .  .  . 
Ferrous  sulphate,  saturated  aqueous  solution  .  . 
Fuchsin,  saturated  alcoholic  solution  ..... 


(6)  Stain.— 

Weigh  out 

Methylene-blue    ..............   \ 

Or  methylene-violet     ............    1  4  grammes 

Or  fuchsin     ................  J 

and  dissolve  in 

Aniline  water,  freshly  saturated  and  filtered    .    .  100  c.c. 


TO   STAIN   SPORES.  91 

METHOD. — 

1.  Prepare  and  fix  films  as  above. 

2.  Pour  the  mordant  on  to  the  film  and  warm  cautiously 
Over  the  flame  till  steam  rises;  keep  the  mordant  gently 
steaming  for  one  minute. 

3.  Wash  well  in  distilled  water  till  no  more  colour  is  dis- 
charged; if  necessary,  wash  carefully  with  absolute  alcohol. 

4.  Filter  a  few  drops  of  the  stain  on  to  the  film,  warm 
as  before,  and  allow  the  steaming  stain  to  act  for  one  minute. 

5.  Wash  well  in  distilled  water. 

6.  Dry  and  mount. 

NOTE. — The  flagella  of  some  organisms  can  be  demon- 
strated better  by  means  of  an  alkaline  stain  or  an  acid  stain 
— a  point  to  be  determined  for  each.  According  to  require- 
ments, therefore,  LofBer  recommends  the  addition  of  sodium 
hydrate,  i  per  cent,  aqueous  solution,  i  c.c. ;  or  an  equal 
quantity  of  an  exactly  comparable  solution  of  sulphuric 
acid. 


To  Stain  Spores. 
1.  Single  Stain.— 

1.  Prepare  cover-slip  film  in  the  usual  way. 

2.  In  fixing,  pass  the  cover-slip  film  fifteen  or  thirty 
times  through  the  flame  instead  of  only  three.     This 
destroys  the  resisting  power  of  the  spore  membrane 
and  allows  the  stain  to  reach  the  interior. 

3.  Stain  in  the  usual  way  with  methylene-blue  or 
fuchsin. 

4.  Wash  in  water. 

5.  Dry  and  mount 
2.  Double  Stain.— 

1.  Prepare  and  fix  film  in  the  usual  way — i.  e.,  pass 
three  times  through  flame  to  fix. 

2.  Cover  the  film  with  hot  carbol-fuchsin  and  hold 
in  the  forceps  above  a  small  flame  until  the  fluid  begins 
to  steam.     Set  the  cover-slip  down  and  allow  it  to 
cool.     Repeat  the  process  when  the  stain  ceases  to 
steam  and  continue  to  repeat  until  the  stain  has  been 
in  contact  with  the  film  for  twenty  minutes.      (This 
stains  both  spores  and  bacteria.) 

3.  Wash  in  water. 


92  STAINING   METHODS. 

4.  Decolourise  in  alcohol,  2  parts;  acetic  acid,  i  per 
cent.,  i  part.     (This  removes  the  stain  from  everything 
but  the  spores.) 

5.  Wash  in  water. 

6.  Mount  the  cover-slip  in  water  and  examine  micro- 
scopically with  the  ^-inch  objective.     (Spores  should 
be  red,  and  the  rest  of  the  film  colourless  or  a  very 
light  pink.)     If  satisfactory,  pass  on  to  section  7;  if 
unsatisfactory,  repeat  steps  2  to  5. 

7.  Counterstain    in    weak    methylene-blue.     (Now 
spores  red,  bacilli  blue.) 

8.  Wash  in  water. 

9.  Dry  and  mount. 

The  spores  of  different  bacilli  differ  greatly  in  their 
resistance  to  decolourising  reagents;  even  the  spores 
of  the  same  species  of  organisms  vary  according  to 
their  age.  Young  spores  are  more  easily  decolourised 
than  those  more  mature. 

Sulphuric  acid,  i  per  cent,  aqueous  solution,  and 
hydrochloric  acid,  0.5  per  cent,  alcoholic  (90  per  cent.) 
solution,  are  useful  decolourising  reagents. 


3.  Muller's  Method.— 

1.  Prepare  and  fix  films  in  the  usual  manner. 

2.  Immerse  in  absolute  alcohol  for  two  minutes,  then  in 
chloroform  for  two  minutes ;  wash  in  water.     This  dissolves 
out  any  fat  or  crystals  that  might  otherwise  retain  the 
" spore"  stain. 

3.  Immerse  in  chromic  acid,  5  per  cent,  aqueous  solution, 
for  one  minute;  wash  in  water. 

4.  Pour  Ziehl's  carbolic  fuchsin  on  the  film,  warm  as  in 
previous  methods,  and  allow  it  to  act  for  ten  minutes. 

5.  Wash  in  water. 

6.  Decolourise  in  sulphuric  acid,  5  per  cent,  aqueous  solu- 
tion, for  five  seconds. 

7.  Wash  in  water. 

8.  Counterstain  with  Kuhne's  carbolic  methylene-blue  for 
one  or  two  minutes. 

9.  Wash  in  water. 

10.  Dry  and  mount. 
(Spores  red,  bacilli  blue.) 


DIFFERENTIAL   METHODS    OF   STAINING.  93 

4.  Abbott's  Method.— 

1.  Prepare  and  fix  films  in  the  usual  manner. 

2.  Pour   I/jffler's   alkaline   methylene-blue   on  the   film; 
warm  cautiously  over  the  flame  till  steam  rises  and  allow 
the  hot  steam  to  act  for  one  to  five  minutes. 

3.  Wash  thoroughly  in  water. 

4.  Decolourise  in  nitric  acid,  2  per  cent,  alcoholic  (80  per 
cent.)  solution. 

5.  Wash  thoroughly  in  water. 

6.  Counterstain  in  eosin,  i  per  cent,  aqueous  solution. 

7.  Wash. 

8.  Dry  and  mount. 
(Spores  blue,  bacilli  red.) 


DIFFERENTIAL  METHODS  OF  STAINING. 

Gram's  Method. — This  method  depends  upon  the 
fact  that  the  protoplasm  of  some  bacteria  permits 
aniline  gentian  violet  and  Lugol's  iodine  solution, 
when  applied  consecutively,  to  enter  into  a  chemical 
combination  which  results  in  the  formation  of  a  new 
blue-black  pigment,  only  very  sparingly  soluble  in 
alcohol.  Such  organisms  are  said  to  "  stain  by  Gram." 

1.  Prepare  a  cover-slip  film  and  fix  in  the  usual  way. 

2.  Stain  in  aniline  gentian  violet  three  to  five  minutes. 

To  prepare  aniline  water,  pour  4  or  5  c.c.  aniline  oil  into 
a  stoppered  bottle  and  add  distilled  water,  100  c.c.  Shake 
vigourously  and  filter  immediately  before  use.  The  excess  of 
oil  sinks  to  the  bottom  of  the  bottle  and  may  be  used  again. 

Filter  as  much  aniline  water  on  to  the  cover-slip  as 
it  will  hold;  then  add  the  smallest  quantity  of  alcoholic 
solution  of  gentian  violet  which  suffices  to  saturate  the 
aniline  water  and  form  a  "bronze  scum"  upon  its 
surface. 

3.  Wash  in  water. 

4.  Treat  with  Lugol's  iodine  solution  until  the  film 
is  black  or  dark  brown. 

To  do  this  treat  with  iodine  solution  for  a  few  seconds, 
wash  in  water,  and  examine  the  film  over  a  piece  of 
white  filter  paper.  Note  the  colour.  Repeat  this 


94  STAINING   METHODS. 

process  until  the  film  ceases  to  darken  with  the  fresh 
application  of  iodine  solution. 

I/ugol's  solution  is  prepared  by  dissolving 

Iodine I  gramme 

Iodine  of  potassium 3  grammes 

In  distilled  water 300  c.c. 

5.  Wash  in  water. 

6.  Wash  with  alcohol  until  no  more  colour  is  dis- 
charged and  the  alcohol  runs  away  clear  and  colourless. 

7.  Wash  in  water. 

8.  Counterstain  very  lightly  with  dilute  eosin,  dilute 
fuchsin,  or  vesuvin. 

NOTE. — This  section  may  be  omitted  when  dealing 
with  films  prepared  from  pure  cultivations. 

9.  Wash  in  water. 

10.  Dry  and  mount. 

Qram=Weigert  Method. — (Also  extremely  useful  for 
sections.) 

1-5.  Proceed  as  for  the  corresponding  sections  of 
Gram's  method  (quod  vide). 

6.  Dry  in  the  air. 

7.  Wash  in  aniline  oil,  i  part,  xylol,  2  parts,  until 
no  more  colour  is  discharged. 

8.  Wash  in  xylol. 

9.  Mount  in  xylol  balsam. 

(Then  fibrin  and  hyaline  tissue  are  stained  deep  blue, 
whilst  bacteria  which  "  stain  Gram"  appear  of  a  deep 
blue- violet  colour.) 

Modified  Qram=Weigert  Method. — (Employed  to 
demonstrate  trichophyta  in  hair.) 

1 .  Soak  the  hairs  in  ether  for  ten  minutes  to  remove 
the  fat. 

2.  Stain  ten  to  sixty  minutes  in  a  tar-like  solution 
of  aniline  gentian  violet  (prepared  by  adding  15  drops 
of  the  alcoholic  solution  of  gentian  violet  to  3  drops 
of  aniline  water). 


TO  DIFFERENTIATE  THE  DIPHTHERIA  BACIIJvUS.     95 

3.  Dry  the  hairs  between  pieces  of  blotting  paper. 

4.  Treat  with  perfectly  fresh  iodine  solution. 

5.  Again  dry  between  blotting  paper. 

6.  Treat  with  aniline  oil.     (If  necessary,  add  a  drop 
or  two  of  nitric  acid  to  the  oil.) 

7.  Again  treat  with  aniline  oil. 

8.  Treat  with  aniline  oil  and  xylol,  equal  parts. 

9.  Clear  with  xylol. 

10.  Mount  in  xylol  balsam. 

To  Differentiate  the  Tubercle  Bacillus  and  Other  Acid= 
fast  Bacilli   (Ziehl-Neelsen's  Method). - 

1.  Smear  a  thin,  even  film  of  the  specimen  on  the 
cover-slip  by  means  of  the  platinum  loop.     (In  the 
case  of  sputum,  if  it  is  a  very  watery  specimen,  allow 
the  film  to  dry,  then  spread  a  second  and  even  a  third 
layer  over  the  first.) 

2.  Fix  by  passing  three  times  through  the  flame. 

3.  Stain  in  hot  carbol-fuchsin    (as  in  staining  for 
spores)  for  five  to  ten  minutes.      (This  stains  every- 
thing on  the  film.) 

4.  Decolourise  by  dipping  in  sulphuric  acid,  25  per 
cent.     (This  removes  stain  from  everything  but  acid- 
fast  bacilli;  e.  g.,  tubercle,  leprosy,  and  smegma  bacilli.) 

5.  Wash  in  water. 

6.  Wash  in  alcohol  till  no  more  colour  is  discharged. 
(This  usually,  but  not  invariably,  removes  the  stain 
from  acid-fast  bacilli  other  than  tubercle;  e.  g.,  smegma 
bacillus.) 

7.  Wash  in  water. 

8.  Counterstain  in  weak    methylene-blue.     (Stains 
non-acid-fast  bacilli,  leucocytes,  epithelial  cells,  etc.) 

9.  Wash  in  water,  dry,  and  mount. 

To  Differentiate  the  Diphtheria  Bacillus  (Neisser's 
Method). - 
Stain  I. — 
Weigh  out 

Methylene-blue I  gramme 


96  STAINING   METHODS. 

and  dissolve  in 

95  per  cent,  alcohol 20  c.c. 

Distilled  water 950    " 

then  add 

Acetic  acid  (glacial) 50    " 

Filter. 
Stain  II. — 
Weigh  out 

Vesuvin 2  grammes 

and  dissolve  in 

Distilled  water,  boiling 1000  c.c. 

Cool  and  filter. 
METHOD.— 

1.  Prepare  and  fix  films  in  the  usual  way. 

2.  Pour  stain  I  on  the  film  and  allow  it  to  act  for 
thirty  seconds. 

3.  Wash  thoroughly  in  water. 

4.  Pour  stain  II  on  to  the  film  and  allow  it  to  act  for 
thirty  seconds. 

5.  Wash  thoroughly  in  water. 

6.  Dry  and  mount. 

NOTE. — The  cultivation  from  which  the  films  are  pre- 
pared must  be  upon  blood-serum  which  has  been  in- 
cubated at  37°  C.  for  from  nine  to  eighteen  hours. 

The  bacilli  are  stained  a  light  brown  by  the  vesuvin, 
which  contrasts  well  with  the  two  or  three  black  spots, 
situated  at  the  poles  and  occasionally  one  in  the  centre 
representing  protoplasmic  aggregations  (?  meta-chro- 
matic  granules)  stained  by  the  acid  methylene-blue. 

To  Demonstrate  the  (?)  Syphilis  Bacillus  (Lustgarten's 
Method).— 

1.  Prepare  and  fix  the  film  in  the  usual  manner. 

2.  Stain  in  aniline  gentian  violet  or  aniline  fuchsin,  twenty- 
four  hours. 

3.  Wash  in  water. 

4.  Place  in   1.5   per  cent,   solution  of  permanganate  of 


TO   DEMONSTRATE   THE    SYPHILIS    BACILLUS.         97 

potash  (a  brown  precipitate  of  oxide  of  manganese  forms), 
three  to  four  seconds. 

5.  Wash  in  watery  solution  of  pure  SO2.     If  not  decolour- 
ised, repeat  sections  4  and  5  until  satisfactory. 

6.  Wash  in  water,  dry,  and  mount. 
Or— 

1.  Prepare  and  fix  the  film  in  the  usual  manner. 

2.  Stain  in  hot  carbol-fuchsin. 

3.  Wash  in  water  to  which  has  been  added  2  or  3  drops 
of  chloride  of  lime. 

4.  Decolourise  in  concentrated  solution  of  chloride  of  iron. 

5.  Wash  thoroughly  in  water. 

6.  Dry  and  mount. 
Syphilis  bacilli  remain  red. 

NOTE. — These  methods  are  equally  applicable  to  tissue 
sections. 


VL  METHODS  OF  DEMONSTRATING 
BACTERIA  IN  TISSUES. 

FOR  bacteriological  purposes,  sections  of  tissues  are 
most  conveniently  prepared  by  either  the  freezing 
method  or  the  paraffin  method. 

The  latter  is  decidedly  preferable,  but  as  it  is  of 
greater  importance  to  demonstrate  the  bacteria,  if  such 
are  present,  than  to  preserve  the  tissue  elements  un- 
altered, the  "frozen"  sections  are  often  of  value. 

Whichever  method  is  selected,  it  is  necessary  to 
take  small  pieces  of  the  tissue  for  sectioning, — 0.5  c.c. 
cubes  when  possible, — not  exceeding  half  a  centimetre 
in  thickness.  Post-mortem  material  should  be  secured 
as  soon  after  the  death  of  the  animal  as  possible. 

The  tissue  is  prepared  for  cutting  by— 

(a)  Fixation;  that  is,  by  causing  the  death  of  the 
cellular  elements  in  such  a  manner  that  they  retain 
their  characteristic  shape  and  form. 

The  fixing  fluids  in  general  use  are :  Absolute  alcohol ; 
corrosive  sublimate,  saturated  aqueous  solution;  cor- 
rosive sublimate,  Lang's  solution  (vide  page  76); 
formaldehyde,  i  per  cent,  aqueous  solution.  (Of 
these,  Lang's  corrosive  sublimate  solution  is  decidedly 
the  best  all-round  " fixative.") 

(6)  Hardening;  that  is,  by  rendering  the  tissue  of 
sufficient  consistency  to  admit  of  thin  slices  or  "  sec- 
tions" being  cut  from  it.  This  is  effected  by  passing 
the  tissue  successively  through  alcohols  of  gradually 
increasing  strength:  30  per  cent,  alcohol,  50  per  cent, 
alcohol,  75  per  cent,  alcohol,  90  per  cent,  alcohol,  recti- 
fied alcohol,  absolute  alcohol. 

In  both  these  processes  a  large  excess  of  fluid  should 

always  be  used. 

98 


FREEZING   METHOD. 


99 


FREEZING    METHOD. 

1.  Fixation.     Place  the  pieces  of  tissue  in  a  wide- 
mouthed  glass  bottle  and  fill  with  absolute  alcohol.  Al- 
low the  tissues  to  remain  therein  for  twenty-four  hours. 

2.  Hardening.     Remove  the  alcohol  (no  longer  abso- 
lute, as  it  has  taken  up  water  from  the  tissues)  from 
the  bottle  and  replace 

it  with  fresh  absolute 
alcohol.  Allow  the  tis- 
sues to  remain  therein 
for  twenty-four  hours. 

NOTE. — If  not  need- 
ed for  cutting  imme- 
diately, the  hardened 
tissues  can  be  stored 
in  50  per  cent,  alco- 
hol. 

3.  Remove  the  alco- 
hol from  the  tissues  by 
soaking  in  water  from 
one    to    two     hours. 
Remove    the    stopper 
from  the  bottle;   rest 
a  glass  funnel  in  the 
open  mouth  and  place 
under  a  tap  of  running 
water.     The  water,  of 

course,  overflows,  but  the  tissues  remain  in  the  bottle. 

4.  Impregnate  the  tissues  with  mucilage  for  twelve 
to  twenty-four  hours,  according  to  size.     Transfer  the 
pieces  of  tissue  to  a  bottle  containing  sterilised  gum 
mixture. 

Formula. — 

Gum  arable 5  grammes 

Saccharose I  gramme 

Boric  acid I        " 

Water  .  .  100  c.c. 


Fig-  53' — Washing  tissues. 


100        TO    DEMONSTRATE    BACTERIA   IN   TISSUES. 

5.  Place  the  tissue  on  the  plate  of  a  freezing  micro- 
tome (Cathcart's  is  perhaps  the  best  form),  cover  and 
surround  with  fresh   gum    mixture;    freeze,  and    cut 
sections. 

6.  Float  the  sections  off  the  knife  into  a  glass  dish 
containing  tepid   water   and   allow   them   to   remain 
therein  for  about  an  hour  to  dissolve  out  the  gum. 

(If  not  required  at  once,  store  in  50  per  cent,  alcohol.) 

7.  Transfer  to  a  glass  capsule  containing  the  selected 
staining  fluid,  by  means  of  a  section  lifter. 

8.  Transfer  the  sections  in  turn  to  a  capsule  con- 
taining absolute  alcohol   (to  dehydrate)   and  to  one 
containing  xylol  or  oil  of  cloves  (to  clear). 

Alternative  Method. — 

8a.  Place  the  stained  section  in  a  dish  of  clean  water 
and  introduce  a  glass  slide  obliquely  beneath  the  sec- 
tion; with  a  mounted  needle  draw  the  section  on  to 
the  slide  and  hold  it  there;  gently  remove  the  slide 
from  the  water,  taking  care  that  any  folds  in  the  sec- 
tion are  floated  out  before  the  slide  is  finally  removed 
from  the  water. 

86.  Drain  away  as  much  water  as  possible  from  the 
section.  Drop  absolute  alcohol  on  to  the  section  from 
a  drop  bottle,  to  dehydrate  it. 

Sc.  Double  a  piece  of  blotting  paper  and  gently  press 
it  on  the  section  to  dry  it. 

Sd.  Drop  on  xylol  to  clear  the  section. 

9.  Place  a  large  drop  of  xylol  balsam  on  the  section 
and  carefully  lower  a  cover-glass  on  to  the  balsam. 

PARAFFIN  METHOD, 

i.  Fixation.  Place  the  pieces  of  tissue,  resting  on 
cotton- wool,  in  a  wide-mouthed  glass  bottle.  Pour  on 
a  sufficient  quantity  of  the  corrosive  sublimate  fixing 
fluid;  allow  the  tissue  to  remain  therein  for  twelve  to 
twenty-four  hours  according  to  size. 


PARAFFIN    METHOD. 


101 


2.  Pour  off  the  fixing  fluid  and  wash  thoroughly  in 
running  water  for  twenty  minutes  to  half  an  hour  to 
remove  the  excess  of  corrosive  sublimate. 

3.  Hardening.     Place  the  tissues  in  each  of  the  fol- 
lowing strengths  of  alcohol  in  turn  for  from  twelve  to 
twenty-four  hours:  50  per  cent.,  75  per  cent.,  90  per 
cent.,  absolute. 

4.  Dehydrate  by  transferring  to  fresh  absolute  alco- 
hol. 

5.  Clear  by  immersing  in  equal  parts  of  absolute  alco- 
hol and  xylol  for  six  to  eighteen  hours ;  then  in  pure 
xylol  for  from  fifteen  minutes  to  six  hours.     The  tissue 
should  not  be  allowed  to  remain  in  the  xylol  longer 
than  is  necessary.     When    "cleared,"  the  tissue  be- 
comes more  or  less  transparent. 


Fig.  54.  —  L- shaped  brass  moulds. 


Fig.  55.— Paraffin  kettle. 


6.  Transfer  the  tissues  to  a  vessel  containing  melted 
paraffin.     Place  this  vessel  in   a  paraffin  water-bath 
regulated  for  2°  C.   above  the  melting-point  of  the 
paraffin  used  and  allow  the  tissues  to  soak  for  some 
six  to  twelve  hours  to  ensure  complete  impregnation. 
The  paraffin  used  should  have  a  melting-point  of  not 
more  than  58°  C.     For  all  ordinary  purposes  54°  C. 
will  be  found  quite  high  enough. 

7.  Imbed  in  fresh  paraffin  in  a  metal  ( or  paper)  mould, 
(a)  Arrange  a  pair  of  L-shaped  pieces  of  metal  on 

a  plate  of  glass  to  form  a  rectangular  trough  (Fig.  54). 


102        TO   DEMONSTRATE   BACTERIA   IN   TISSUES. 

(b)  Pour  fresh  melted  paraffin  into  the  mould  from  a 
special  vessel  (Fig.  55). 

(c)  Lift  the  piece  of  tissue  from  the  paraffin  bath 
and  arrange  it  in  the  mould. 

(d)  Blow  gently  on  the  surface  of  the  paraffin  in  the 
mould,  and  as  soon  as  a  film  of  solid  paraffin  has  formed, 
carefully  lift  the  glass  plate  on  which  the  mould  is  set 
and  lower  plate  and  mould  together  into  a  basin  of 
cold  water. 

(e)  When  the  block  is  cold,  break  off  the  metal  L's; 
trim  off  the  excess  of  paraffin  from  around  the  tissue 
with  a  knife,  taking  care  to  retain  the  rectangular 
shape,  and  store  the  block  in  a  pill-box. 

When  several  pieces  of  tissue  have  to  be  imbedded 
at  one  time,  shapes  of  copper,  10 
cm.,  5  cm.,  and  2.5  cm.  square  re- 
spectively, and  0.75  cm.  deep  (Fig. 
56)  will  be  found  extremely  useful. 
These  placed  upon  plates  of  glass 
replace  the  pair  of  L's  in  the  above 

Fig.  56.— Paraffin 

'   mould.  process. 

8.    Cement    the    block    on    the 

carrier  of  a  "paraffin"  microtome  (the  Minot,  the 
Jung,  or  the  Cambridge  Rocker)  with  a  little  melted 
paraffin.  Greater  security  is  obtained  if  the  paraffin 
around  the  base  of  the  block  is  melted  by  means  of  a 
hot  metal  or  glass  rod. 

9.  Cut  sections — thin,  and  if  possible  in  ribbands. 
Mounting  Paraffin  Sections. — 

1.  Place  a  large  drop  of  30  per  cent,   alcohol  on 
the  centre  of  a  slide  (or  cover-slip)  and  float  the  sec- 
tion on    to   the  surface  of  the  drop,  from  a  section 
lifter. 

2.  Hold  the  slide  in  the  fingers  of  one  hand  and  warm 
cautiously  over  the  flame  of  a  Bunsen  burner,  touch- 
ing the  under  surface  of  the  glass  from  time  to  time 
on  the  back  of  the  other  hand.     As  soon  as  the  slide 


STAINING   PARAFFIN   SECTIONS. 


I03 


ig.  57. — Section  rack. 


feels  distinctly  warm  to  the  skin,  the  paraffin  section 
will  flatten  out  and  all  wrinkles  disappear. 

(The  slide  with  the  section  floating  on  it  may  be 
rested  on  the  top  of  the  paraffin  bath  for  two  or  three 
minutes,  instead  of  warm- 
ing over  the  flame  as  here 
described.) 

3.  Cautiously    tilt    up 
the  slide  and  blot  off  the 
excess  of  spirit  with  blot- 
ting   paper,    leaving    the 
section    attached    to    the 
centre  of  the  slide. 

4.  Place  the   slide   in  a 
wire  rack  (Fig.  57),  section 
downwards,  in  the  "hot" 
incubator   for    twelve    to 
twenty-four    hours.       At 

the  end  of  this  time  the  section  is  firmly  adherent  to 
the  glass,  and  is  treated  during  the  subsequent  steps 
as  a  "fixed"  cover-glass  film  preparation. 

NoTS. — If  large,  thick  sections  have  to  be  manipulated, 
or  if  time  is  of  importance,  it  is  often  advisable  to  add  a 
trace  of  Mayer's  albumin  to  the  alcohol  before  floating  out 
the  section.  If  this  substance  is  employed,  a  sojourn  of 
twenty  minutes  to  half  an  hour  in  the  "hot"  incubator  will 
be  found  ample  to  ensure  firm  adhesion  of  the  section  to  the 
slide.  Mayer's  albumin  is  prepared  as  follows: 

Weigh  out 

Salicylate  of  soda I  gramme 

and  dissolve  in 

Glycerine 50  c.c. 

Add 

White  of  egg 50    " 

Mix  thoroughly  by  means  of  an  egg  whisk. 
Filter  into  a  clean  bottle. 

Staining  Paraffin  Sections. — 

i.  Warm  paraffin  section  over  the  Bunsen  flame  to 


104       TO   DEMONSTRATE    BACTERIA   IN   TISSUES. 

soften  (but  not  to  melt)  the  paraffin,  then  dissolve  out 
the  wax  with  xylol. 

2.  Remove  xylol  by  flushing  the  section  with  alcohol. 

3.  If  the  tissue  was  originally  "fixed"  in  a  corrosive 
sublimate  solution,  the  section  must  now  be  treated 
with  lyUgol's  iodine  solution  for  five  minutes. 

4.  Stain  deeply,  if  using  a  single  stain,  as  the  sub- 
sequent processes  decolourise. 

5.  Wash  in  water,  decolourise  if  necessary. 

6.  Flood  with  several  changes  of  absolute  alcohol  to 
dehydrate  the  section. 

7.  Clear  in  xylol.     (Oil  of  cloves  is  not  usually  em- 
ployed, as  it  decolourises  the  section.) 

8.  Mount  in  xylol  balsam. 


SPECIAL  STAINING  METHODS  FOR  SECTIONS. 

Double=staining  Carmine  and  Gram=Weigert.— 

1.  Prepare    the    sec- 
tion    for     staining     as 
above,  sections  i  to  3. 

2.  Stain    in    lithium 
carmine     ( Orth's )     or 
picrocarmine     for     ten 
to  thirty  minutes  in  a 
porcelain   staining    pot 
(Fig.  58). 

3.  Wash  in  picric  acid 
solution    until    yellow. 

At  this  stage  cell  nuclei  are  red,  protoplasm  is  yellow, 
and  bacteria  are  colourless. 

Picric  acid  solution  is  prepared  by  mixing 

Picric  acid,  saturated  aqueous  solution      .    .    .    .    40  c.c. 

Hydrochloric  acid i    " 

Alcohol  (90  per  cent.) 160  " 

4.  Wash  in  water. 

5.  Wash  in  alcohol. 


Fig.  58. — Staining  pot. 


TO   DEMONSTRATE   CAPSULES.  105 

6.  Stain  in  aniline  gentian  violet. 

7.  Wash  in  iodine  solution  till  dark  brown  or  black. 

8.  Wash  in  water. 

9.  Dip  in  absolute  alcohol  for  a  second. 

10.  Decolourise  with  aniline  oil  till  no  more  colour 
is  discharged. 

11.  Wash  with  aniline  oil,  2  parts,  xylol,  i  part. 

12.  Clear  with  xylol. 

13.  Mount  in  xylol  balsam. 

Alternative  Qram=Weigert  Method  for  Sections. — 

1 .  Fix  paraffin  section  on  slide  and  prepare  for  stain- 
ing in  the  usual  manner. 

2.  Stain  in  alum  carmine  for  about  fifteen  minutes. 

3.  Wash  thoroughly  in  water. 

4.  Filter  aniline  gentian  violet  solution  on  to  the 
section  on  the  slide  and  allow  to  stain  about  twenty- 
five  minutes. 

5.  Wash  thoroughly  in  water. 

6.  Treat  with  Lugol's  iodine  until  section  ceases  to 
become  any  blacker. 

7.  Wash  thoroughly  in  water. 

8.  Treat  with  a  mixture  of  equal  parts  of  aniline 
oil  and  xylol  until  no  more  colour  comes  away. 

9.  Wash  thoroughly  with  xylol. 

10.  Decolourise  and  dehydrate  rapidly  with  abso- 
lute alcohol  until  there  remains  only  a    very  faint 
bluish  tint. 

1 1 .  Clear  with  xylol. 

12.  Mount  in  xylol  balsam. 
To  Demonstrate  Capsules.— 

1.  MacConkey's    Method. — Stain    precisely    as    for 
cover-slip  films. 

2.  Friedldnder's  Method. — 
Stain.— 

Gentian  violet,  saturated  alcoholic  solution  .    .    .     50  c.c. 

Acetic  acid,  glacial 10    " 

Distilled  water     ,  .  100   " 


106        TO   DEMONSTRATE   BACTERIA   IN   TISSUES. 

METHOD. — 

1.  Prepare  the  sections  for  staining,  secundum  artem. 

2.  Stain  sections  in  the  warm  (e.  g,,  in  the  hot  incubator) 
for  twenty-four  hours. 

3.  Wash  with  water. 

4.  Decolourise  lightly  with  acetic  acid,  i  per  cent. 

5.  Dehydrate  rapidly  with  absolute  alcohol. 

6.  Clear  with  xylol. 

7.  Mount  in  xylol  balsam. 

To  Demonstrate  Acid=fast  Bacilli.— 

1.  Prepare  the  sections  for  staining  in  the  usual 
way. 

2.  Stain  with  haematin  solution  ten  to  twenty  sec- 
onds, to  obtain  a  pure  nuclear  stain ;  then  wash  in  water. 

3.  Stain    with    carbolic   fuchsin    twenty    to    thirty 
minutes  at  47°  C.;  then  wash  in  water. 

4.  Treat  with   aniline   hydrochlorate,    2   per   cent, 
aqueous  solution,  for  two  to  five  seconds. 

5.  Decolourise  in  75  per  cent,  alcohol  till  section 
appears  free  from  stain — fifteen  to  thirty  minutes. 

6.  Dehydrate  with  absolute  alcohol. 

7.  Clear  very  rapidly  with  xylol. 

8.  Mount  in  xylol  balsam. 


VH.  CLASSIFICATION  OF  FUNGI. 

FUNGI  are  divided  into: 

1.  Hymenomycetes  (including  the  mushrooms,  etc.). 

2.  Hyphomycetes  (moulds). 

3.  Blastomycetes  (yeasts  and  torulse). 

4.  Schizomycetes  (bacteria). 

NOTE. — Formerly  myxomycetes  were  included  in 
the  fungi ;  they  are  now  recognised  as  belonging  to  the 
animal  kingdom,  and  are  termed  "mycetozoa." 


MORPHOLOGY  OF  THE  HYPHOMYCETES. 

At  the  commencement  of  his  studies,  the  attention 
of  the  student  is  directed  to  the  various  non-pathogenic 
moulds  and  yeasts,  not  only  that  he  may  gain  the 
necessary  technique  whilst  handling  cultivations  of 
harmless  organisms,  but  also  because  these  very  species 
are  amongst  the  commonest  of  those  that  may  acci- 
dentally contaminate  his  future  preparations. 

The  hyphomycetes  are  composed  of  a  mycelium  of 
short  jointed  rods  or  "hyphae"  springing  from  an  axis 
or  germinal  tube  which  develops  from  the  spore.  Hy- 
phae  are — 

(a)  Nutritive  or  submerged. 

(6)  Reproductive  or  aerial. 

The  protoplasm  of  these  cells  contains  granules, 
pigment,  oil  globules,  and  sometimes  crystals  of  cal- 
cium oxalate. 

Reproduction. — Apical     spore    formation — asexual; 

zoospores — sexual. 

Mucorinse. — Mucor  (Fig.  59). — Note  the  branching 
filaments— "mycelium"  (a),  "hyphse"  (6). 

Note  the  asexual  reproduction.. 

107 


io8 


CLASSIFICATION   OF   FUNGI. 


1 .  A  filament  grows  upwards.     At  its  apex  a  septum 
forms,  then  a  globular  swelling  appears — "  sporagium" 
(d).     This  possesses  a  definite  membrane. 

2.  From  the  septum  grows  a  club-shaped  mass  of 
protoplasm — "columella"  (c). 


Fig-  59- — Mucor  mucedo. 


Fig.  60. — Aspergillus. 


3.  The  rest  of  the  contained  protoplasm  breaks  up 
into  "swarm  spores"  (e). 

Finally  the  membrane  ruptures  and  spores  escape. 
Perisporaceae. — Aspergillus     (Fig.     60). — Note    the 
branching  filaments — "mycelium"  (a). 

Note    the    asexual 
reproduction.    - 

1.  A  filament    (b) 
grows    upwards,    its 
termination  becomes 
clubbed ;       on       the 
clubbed       extremity 
flask-shaped  cells  ap- 
pear —  "  sterigmata" 

Fig.  61. — Penicillium.  (c). 

2.  At  free    end   of 

each  sterigma  is  formed  an  oval  body — a   spore  or 
"gonidium"  (d),  which,  when  ripe,  is  thrown  off  from 
the  sterigma. 


MORPHOLOGY    OF   THE    B^ASTOMYCETES.          109 

Penicillium  (Fig.  6 1 ) . — Note  the  branching  filaments 
— " mycelium"  (a)   (frequently  containing  globules). 
Note  the  asexual  reproduction. 

1 .  A  filament  grows  upwards — ' '  goniodophore' '  ( b ) — 
and  its   apex    divides    up    into    several    branches — 
"basidia"(c). 

2.  At  the  apex  of  each  branch  a  flask-shaped  cell, 
"sterigma"  (d),  appears. 

3.  At  the  apex  of  each  sterigma  appears  a  row  of 
oval  cells — "spores"  or  "conidia"  (e).     These,  when 
ripe,  are  cast  off  from  the  sterigmata. 

Ascomycetse. — O'idium   (Fig.    62). — (This  family  is 
perhaps   as    nearly    re- 
lated to  the  blastomy- 
cetes  as  it  is  to  the  hy- 
phomycetes.) 

Note  the  branching 
filaments  —  "pseudo- 
mycelium  "  ( a ) .  Here 
and  there  filaments  are 
broken  up  at  their  ends 
into  oval  or  rod-shaped  Fig'  62'~ Oidium- 

segments,   "oi'dia,"  and  behave  as  spores. 

Note  the  asexual  reproduction.  From  the  pseudo- 
mycelium  arise  true  hyphae  (b),  each  of  which  in  turn 
ends  in  a  chain  of  spores  (c). 


MORPHOLOGY  OF  THE  BLASTOMYCETES. 

The  blastomycetes  are  composed  of  spherical  or  oval 
cells  (8  to  9.5  /i  in  diameter),  which,  when  rapidly 
multiplying  by  budding,  may  form  a  spurious  myce- 
lium. A  thin  cell-wall  encloses  the  granular  proto- 
plasm, in  which  vacuoles  and  sometimes  a  nucleus 
may  be  noted.  This  latter  is  best  seen  when  stained 
with  osmic  acid  or  haematoxylin. 

During  their  growth  and  multiplication  the  blasto- 


no 


CLASSIFICATION   OF   FUNGI. 


mycetes  split  up  solutions  containing  sugar  into  alcohol 
and  CO2. 

Reproduction. — Budding,  ascospores — asexual. 

(Torulae,  whilst  resembling  yeasts  in  almost  every 
other  respect,  never  form  spores.) 

Saccharomyces  (Fig.  63). — Note  the  round  or  oval 
cells  of  granular  protoplasm  (a)  containing  solid  par- 
ticles and  vacuoles  (c),  and  surrounded  by  a  definite 
envelope. 

Note  the  asexual  reproduction. 

i.  "Gemmation" — that  is,  the  budding  out  of 
daughter  cells  (b)  from  various  parts  of  the  gradually 
enlarging  mother  cell.  These  are  eventually  cast  off 


Fig.  63. — Saccharomyces 
with  ascospores. 


Fig.  64. — Torula. 


and  in  turn  become  mother  cells  and  form  fresh  groups 
of  buds. 

2.  Spore  formation — "ascospores"  (e).  These  are 
formed  at  definite  temperatures  and  within  well-de- 
fined periods;  e.  g.,  Saccharomyces  cerevisiae,  thirty 
hours  at  25°  to  37°  C.,  or  ten  days  at  12°  C. 

Torulae  (Fig.  64). — Note  the  points  in  morphology, 
etc.,  as  above. 

Note  the  absence  of  ascospore  formation. 


VIIL  SCHIZOMYCETES. 

Classification  and  Morphology. — Bacteria  are  often 
classified,  in  general  terms,  according  to  their  life 
functions,  into — 

Saprogenic,  or  putrefactive  bacteria; 
Zymogenic,  or  fermentative  bacteria; 
Pathogenic,  or  disease-producing  bacteria; 
or  according  to  their  metabolic  products  into — 

Chromogenic,  or  pigment-producing  bacteria; 
Photogenic,  or  light-producing  bacteria; 
Aerogenic,  or  gas-producing  bacteria; 
and  so  on. 

Such  broad  groupings  as  these  have,  however,  but 
little  practical  value  when  applied  to  the  systematic 
study  of  the  fission  fungi. 

On  the  other  hand,  no  really  scientific  classification 
of  the  schizomycetes  has  yet  been  drawn  up,  and  the 
varying  morphological  appearances  of  the  members 
of  the  family  are  still  utilised  as  a  basis  for  classifica- 
tion, as  under— 

i.  Micrococci,  or  Cocci  (Fig.  65). — Rounded  or  oval 
cells,  subdivided  according  to  the  arrangement  of  the 
individuals  after  fission,  into — 

Diplococci  and  Streptococci,  where  division  takes 
place  in  one  plane  only,  and  the  individuals  remain 
attached  (a)  in  pairs  or  (b)  in  chains. 

Tetrads,  Merismopedia,  or  Pediococci,  where  divi- 
sion takes  place  alternately  in  two  planes  at  right 
angles  to  each  other,  and  the  individuals  remain  at- 
tached in  flat  tablets  of  four,  or  its  multiples. 

Sarcinae,  where  division  takes  place  in  three  planes 
successively,  and  the  individuals  remain  attached  in 
cubical  packets  of  eight  and  its  multiples. 

in 


112 


SCHIZOMYCETES. 


Staphylococci,  where  division  takes  place  in  three 
planes,  but  with  no  definite  sequence;  consequently 
the  individuals  remain  attached  in  pairs,  short  chains, 
plates  of  four,  cubical  packets  of  eight,  and  irregular 
masses  containing  numerous  cocci. 


Fig.  65. — Types  of  bacteria — cocci :  I,  Diagram  of  sphere  indicating  planes 
of  fission;  2,  diplococci ;  3,  streptococci;  4,  tetrads;  5,  sarcinse ;  6,  staphy- 
lococci. 


2.  Bacilli  (Fig.  66,  i  to  3). — Rod-shaped  cells.  A 
bacillus,  however  short,  can  usually  be  distinguished 
from  a  coccus  in  that  two  sides  are  parallel.  Some 
bacilli  after  fission  retain  a  characteristic  arrangement 
and  may  be  spoken  of  as  diplobacilli  or  streptobacilli. 


Fig.   66. — Types  of  bacteria — bacilli,  etc.  :   I,  Bacilli ;  2,  diplobacilli ;  3, 
streptobacilli  ;  4,  spirilla ;  5,  vibrios ;  6,  spirochaetae. 


(Leptothrix  is  a  term  that  in  the  past  has  been  loosely 
used  to  signify  a  long  thread,  but  is  now  restricted  to 
such  forms  as  belong  to  the  leptothriciae  (vide  page 


3.  Spirilla  (Fig.  66,  4  to  6).  —  Curved  and  twisted 
filaments.     Classified,  according  to  shape,  into  — 


ANATOMY.  113 

Spirillum. 

Vibrio  (comma). 

Spirochaeta. 

Higher  forms  of  bacteria  are  also  met  with,  which 
possess  the  following  characteristics :  They  are  attached, 
unbranched,  filamentous  forms,  showing — 

(a)  Differentiation  between  base  and  apex; 

(b)  Growth  apparently  apical; 

(c)  Exaggerated  pleomorphism ; 

(d)  "Pseudo-branching"  from  apposition  of  cells; 
and  are  classified  into — 

1 .  Beggiotoa.   1  Free  swimming  forms,  which  contain 

2.  Thiothrix.    /      sulphur  granules. 

3.  Crenothrix.  -\  ^*        £  .    .        ,  - 

These  forms  do  not  contain  sulphur 

4.  Cladothnx.  >  , 

.    .      (      granules. 

5.  Leptothnx.  ) 

6.  Strep tothrix.      A    group    which    exhibits    true 
branching,  and  contains  some  pathogenic  species. 

The  morphology  of  the  same  bacterium  may  vary 
greatly  under  different  conditions  of  growth  as  to — 

1.  The  composition,  reaction,  etc.,  of  the  nutrient 
medium. 

2.  The  atmosphere  in  which  it  is  cultivated. 

3.  The  temperature  at  which  it  is  incubated. 

4.  Exposure  to  or  protection  from  light. 

For  example,  under  one  set  of  conditions  the  exami- 
nation of  a  pure  cultivation  of  a  bacillus  may  show  a 
short  oval  rod  as  the  predominant  form,  whilst  another 
culture  of  the  same  bacillus,  but  grown  under  different 
conditions,  may  consist  almost  entirely  of  long  fila- 
ments or  threads,  a  condition  known  as ' '  pleomorphism. ' " 


ANATOMY. 

i.  Capsule  (Fig.  67,  b). — A  gelatinous  envelope 
(probably  akin  to  mucin  in  composition)  surrounding 
each  individual  organism,  and  preventing  absolute  con- 


SCHIZOMYCETES. 


Fig.  67. — Structure  of  bacteria. 


tact  between  any  two.  In  some  species  the  capsule  is 
well  marked,  but  it  cannot  be  demonstrated  in  all.  In 
very  well  marked  cases  of  gelatinisation  of  the  cell  wall, 

the  individual  cells  are 
cemented  together  in  a 
coherent  mass,  to  which 
the  term  "zoogloea"  is 
applied.  In  some  species 
colouring  matter  or  ferric 
oxide  is  stored  in  the 
capsule. 

2.  Cell  Watt  (Fig.  6?,  c). 
— A  protective  differen- 
tiation of  the  outer  layer 
of  the  cell  protoplasm; 
difficult  to  demonstrate, 
but  treatment  with  io- 
dine or  salt  solution 
sometimes  causes  shrinkage  of  the  cell  contents — "  plas- 
molysis" — and  so  renders  the  cell  wall  apparent  (e.  g.y 
B.  megatherium)  in  the  manner  shown  in  figure  68. 
Stained  bacilli,  when  examined  with 
polarising  microscope,  often  show  a 
doubly  refractile  cell  wall  (e.  g.,  B. 
tuberculosis  and  B.  anthracis). 

In  some  of  the  higher  bacteria  the 
cell  wall  exhibits  this  differentia- 
tion to  a  marked  degree  and  forms 
a  hard  sheath  within  which  the  cell 

Fig.  68. — Plasmolysis. 

protoplasm  is  freely  movable;  and 

during  the  process  of  reproduction  the  cell  protoplasm 

may  be  extruded,  leaving  the  empty  tube  unaltered  in 

shape. 

3.  Cell  Contents. — Protoplasm  (mycoprotein)  con- 
tains a  high  percentage  of  nitrogen,  but  is  said  to  differ 
from  proteid  in  that  it  is  not  precipitated  by  C2HfiO. 
It  is  usually  homogeneous  in  appearance — sometimes 


1 1 5 

granular — and  may  contain  oil  globules  or  sap  vacuoles 
(Fig.  67,  d),  chromatin  granules,  and  even  sulphur 
granules.  Sap  vacuoles  must  be  distinguished  from 
spores,  on  the  one  hand,  and  the  vacuolated  appear- 
ance due  to  plasmolysis,  on  the  other. 

The  cell  contents  may  sometimes  be  differentiated 
into  a  parietal  layer,  and  a  central  body  (e.  g.,  beg- 
giotoa)  when  stained  by  haematoxylin. 

4.  Nucleus. — This  structure  has  not  been  conclu- 
sively proved  to  exist,  but  in  some  bacteria  denser 
masses  of  protoplasm  situated  at  the  poles  and  ex- 
hibiting a  more  marked  affinity  than  the  rest  of  the  cell 
protoplasm  for  aniline  dyes  have  been  observed.     These 
are  termed  polar  granules  or  Polkorner  (Fig.   67,   e). 
Occasionally  these  aggregations  of   protoplasm  alter 
the  colour  of  the  dye  they  take  up.     They  are  then 
known  as  metachromatic  bodies  or  Ernst' schen  Korner. 

5.  Flagella  (Organs  of  Locomotion,  Fig.  67,  a).— 
These  are  gelatinous  elongations  of  the  cell  protoplasm 
(or  more  probably  of  the  capsule),  occurring  either  at 
one    pole,   at   both  poles,  or 

scattered  around  the  entire 
periphery.  Flagella  are  not 
pseudopodia.  The  possession 
of  flagella  was  at  one  time 
suggested  as  a  basis  for  a  sys- 
tem of  classification,  when  the 
following  types  of  ciliation  Fig.  69._Types  of  ciliavtion. 
were  differentiated: 

1.  Polar:  (a)  Monotrichous  (a  single  flagellum  situ- 
ated at  one  pole;  e.  g.,  B.  pyocyaneus). 

(b)  Amphitrichous  (a  single  flagellum  at  each  pole; 
e.  g.,  Spirillum  volutans). 

(c)  Lophotrichous  (a  tuft  or  bunch  of  flagella -situated 
at  each  pole;  e.  g.,  B.  cyanogenus). 

2.  Diffuse :  Peritrichous  (flagella  scattered  around  the 
entire  periphery;  e.  g.,  B.  typhosus). 


1 1 6  SCHIZOMYCETES. 

PHYSIOLOGY. 

Reproduction. — Active  Stage. — Vegetative,  i.  e.,  by 
the  division  of  cells,  or  "fission." 

1.  The  cell  becomes  elongated  and  the  protoplasm 
aggregated  at  opposite  poles. 

2.  A  circular  constriction   of   the   organism  takes 
place  midway  between  these  aggregations,  and  a  sep- 
tum is  formed  in  the  interior  of  the  cell  at  right  angles 
to  its  length. 

3.  The  division  deepens,  the  septum  divides  into  two 
lamellae,  and  finally  two  cells  are  formed. 

4.  The  daughter  cells  may  remain  united  by  the 
gelatinous  envelope  for  a  variable  time.     Eventually 
they  separate  and  themselves  subdivide. 


Fig.  70. — Fission  of  cocci.  Fig.  71. — Fission  of  bacteria. 

Cultures  on  artificial  media,  after  growing  in  the 
same  medium  for  some  time, — i.  e.,  when  the  pabulum 
is  exhausted, — show  "involution  forms "  (Fig.  72),  well 
exemplified  in  cultures  of  B.  pestis  on  agar  two  days 
old,  B.  diphtherias  on  potato  four  to  six  days  old. 

They  are  of  two  classes,  viz.: 

(a)  Involution  forms  characterised  by  alterations  of 
shape.     (Not  necessarily  dead.) 

(b)  Involution  forms  characterised  by  loss  of  staining 
power.     (Always  dead.) 

Resting  Stage. — Spore  Formation. — Conditions  in- 
fluencing spore  formation:  In  an  old  culture  nothing 
may  be  left  but  spores.  It  used  to  be  supposed  that 
spores  were  always  formed  when 

(a)  The  supply  of  nutrient  was  exhausted. 


PHYSIOLOGY.  117 

(6)  The  medium  became  toxic  from  the  accumula- 
tion of  metabolic  products. 

(c)  The  environment  became  unfavourable*  e.  g.t 
change  of  temperature. 

So  that  the  species  might  not 
become  extinct.  This  is  not 
altogether  correct;  e.  g.,  the 
temperature  at  which  spores  are 
best  formed  is  constant  for  each 
bacterium,  but  varies  with  dif- 
ferent species;  again,  aerobes 
require  oxygen  for  sporulation, 
but  anaerobes  will  not  spore  in 
its  presence. 

(A)  Arthrogenous :  Noted  only 
in  the  micrococci.    One  complete 

element  resulting  from  ordinary      Fis-  72-— involution  forms. 

fission  becomes  differentiated  for 

the  purpose,  enlarges,  and  develops  a  dense  cell  wall. 

One  or  more  of  the  cells  in  a  series  may  undergo  this 

alteration. 

This  process  is  probably  not  real  spore  formation, 
but  merely  relative  increase  of  resistance.  These  so- 
called  arthrospores  have  never  been  observed  to  ''ger- 
minate," nor  is  their  resistance  very  marked,  as  they 
fail  to  initiate  new  cultures,  after  having  been  exposed 
to  a  temperature  of  80°  C.  for  ten  minutes. 

(B)  Endogenous:  The  cell  protoplasm  becomes  dif- 
ferentiated and  condensed  into  a  spherical  or  oval 
mass  (very  rarely  cylindrical).     After  further  contrac- 
tion the  outer  layers  of  the  mass  become  still  more 
highly  differentiated  and  form  a  distinct  spore  mem- 
brane, and  the  spore  itself  is  now  highly  refractile. 
It  has  been  suggested,  and  apparently  on  good  grounds, 
that  the  spore  membrane  consists  of  two  layers,  the 
exosporium  and  the  endosporium.     Each  cell  forms 
one  spore  only,  usually  in  the  middle,  occasionally  at 


a         b  c  d        e 

IW? 


Il8  SCHIZOMYCETES. 

one  end  (four  exceptions,  however,  are  recorded;  e.  g., 
B.  inflatus).  The  shape  of  the  parent  cell  may  be  un- 
altered, as  in  the  anthrax  bacillus,  or  altered,  as  in  the 
tetanus  bacillus,  and  these  points  serve  as  the  basis 
for  a  classification  of  spore-bearing  bacilli,  as  follows : 

(A)  Cell  body  of  the  parent 
bacillus    unaltered    in    shape 
(Fig.  73,  a). 

(B)  Cell  body  of  the  parent 
bacillus  altered  in  shape. 

Fig-  73-— Types  of  spore-bear-  _.    r 

ing  bacilli.  i.  tlostr^d^um  (Fig.  73,  b): 

Rod  swollen  at  the  centre  and 

attenuated  at  the  poles;  spindle  shape;  e.  g.,  B.  buty- 
ricus. 

2.  Cuneate  (Fig.  73,  c) :  Rods  swollen  slightly  at  one 
pole  and  more  or  less  pointed  at  the  other;  wedge- 
shaped. 

3.  Clavate  (Fig.  73,  d) :  Rods  swollen  at  one  pole  and 
cylindrical  (unaltered)  at  the  other;   keyhole-shaped; 
e.  g.,  B.  chauvei. 

4.  Capitate  (Fig.  73,  e) :  Rods  with  a  spherical  en- 
largement   at   one   pole;    drumstick-shaped;  e.  g.,  B. 
tetani. 

The  endospores  remain  within  the  parent  cell  for  a 
variable  time  (in  one  case  it  is  stated  that  germination 
of  the  spore  occurs  within  the  interior  of  the  parent 
cell — "endo-germination"),  but  are  eventually  set  free, 
as  a  result  of  the  swelling  up  and  solution  of  the  cell 
membrane  of  the  parent  bacillus  in  the  surrounding 
liquid,  or  of  the  rupture  of  that  membrane.  They 
then  present  the  following  characteristics: 

1 .  Well-formed,  dense  cell  membranes,  which  renders 
them  extremely  difficult  to  stain,  but  when  once  stained 
equally  difficult  to  decolourise. 

2.  High  refractility,  which  distinguished  them  from 
vacuoles. 

3.  Higher  resistance  than  the  parent  organism  to 


GERMINATION.  119 

such  lethal   agents   as   heat,    desiccation,   starvation, 
time,  etc.,  this  resistance  being  due  to 

(a)  Low  water  contents  of  plasma        . 

/IN  T        i  j     ^  °i  the  spore 

(b)  Low  heat-conducting  power 

;  ; '  ,  M.  membrane. 

(c)  Low  permeability 

This  resistance  varies  somewhat  with  the  particular 
species, — e.  g.,  some  spores  may  resist  boiling  for  a  few 
minutes, — but  practically  all  are  killed  if  the  boiling  is 
continued  for  ten  minutes. 

Germination. — When  transplanted  to  suitable  media 
and  placed  under  favourable  conditions,  the  spores 
germinate,  usually  within  twenty-four  to  thirty-six 
hours,  and  successively  undergo  the  following  changes : 

1.  Swell  up  slowly  and  enlarge,   through  the  ab- 
sorption of  water. 

2.  Lose  their  refrangibility. 

3.  At  this  stage  one  of  three  processes  (but  the  par- 
ticular process  is  always  constant  for  the  same  species) 
may  be  observed: 

(a)  The  spore  grows  out  into  the  new  bacillus  without 
discarding  the  spore  membrane  (which  in  this  case 
now  becomes  the  cell  membrane);  e.  g.,  B.  lepto- 
sporus. 

(6)  It  loses  its  spore  membrane  by  solution;  e.  g.t  B. 
anthracis. 

(c)  It  loses  its  spore  membrane  by  rupture. 

In  this  process  the  rupture  may  be  either  polar,  and 
taking  place  at  one  pole  only  (e.  g.,  B.  butyricus)  or 
occurring  at  both  poles  (e.  g.,  B.  sessile),  or  equatorial; 
e.  g.,  B.  subtilis. 

In  those  cases  where  the  spore  membrane  is  discarded 
the  cell  membrane  of  the  new  bacillus  may  either  be 
formed  from — 

(a)  The  inner  layer  of  the  spore  membrane,  which 
has  undergone  a  preliminary  splitting  into  parietal 
and  visceral  layers;  e.  g.,  B.  butyricus. 

(6)  The  outer  layers  of  the  cell  protoplasm,  which 


I2O 


SCHIZOMYCETES. 


become  differentiated  for  that  purpose;  e.  g.,  B.  mega- 
therium. 

The  new  bacillus  now  increases  in  size,  elongates, 


O  G 


Fig.  74- 


Fig.  75- 


oO 


Fig.  76. 


•^^^ 

O 


o  0® 


Fig.  77. 


0 


Fig.  78. 


Figs-  74,  75,  76,  77,  78.— Types  of  germination  :     Fig.  74,  simple ;  Fig.  75, 
solution;  Fig.  76,  polar;  Fig.  77,  bipolar;  Fig.  78,  equatorial. 


FOOD   STUFFS.  121 

and  takes  on  a  vegetative  growth, — i.  e.,  undergoes 
fission, — the  bacilli  resulting  from  which  may  in  their 
turn  give  rise  to  spores. 

Food  Stuffs. — i.  Organic  Foods.—- 

(a)  The  pure  parasites  (e.  g.,  B.  leprae)  will  not  live 
outside  the  living  body. 

(6)  Both  saprophytic  and  facultative  parasitic  bac- 
teria agree  in  requiring  non-concentrated  food. 

(c)  The  facultative  parasites  need  highly  organised 
foods;  e.  g.,  proteids  or  other  sources  of  nitrogen  and 
carbon,  and  salts. 

(d)  The  saprophytic  bacteria  are  more  easily  culti- 
vated; e.  g., 

1.  Some  bacteria  will  grow  in  almost  pure  distilled 
water. 

2.  Some  bacteria  will  grow  in  pure  solutions  of  the 
carbohydrates . 

2.  Water  is  absolutely  essential  to  the  growth  of 
bacteria. 

Food  of  a  definite  reaction  is  needed  for  the  growth 
of  bacteria.  As  a  general  rule  growth  is  most  active 
in  media  which  react  slightly  acid  to  phenolphthalein 
— that  is,  neutral  or  faintly  alkaline  to  litmus.  Mould 
growth,  on  the  other  hand,  is  most  vigourous  in  media 
that  are  strongly  acid  to  phenolphthalein. 

Environment. — The  influence  of  physical  agents 
upon  bacterial  life  and  growth  is  strongly  marked. 

i.  Atmosphere. — The  presence  of  oxygen  is  necessary 
for  the  growth  of  some  bacteria,  and  death  follows 
when  the  supply  is  cut  off.  Such  organisms  are  termed 
obligate  aerobes. 

Some  bacteria  appear  to  thrive  equally  well  whether 
supplied  with  or  deprived  of  oxygen.  These  are  termed 
facultative  anaerobes. 

A  third  class  will  only  live  and  multiply  when  the 
access  of  free  oxygen  is  completely  excluded.  These 
are  termed  obligate  anaerobes. 


122  SCHIZOMYCETES. 

2.  Temperature. — Practically    no    bacterial    growth 
occurs  below  5°  C.,  and  very  little  above  40°  C.     30°  C. 
to  37°  C.  is  the  most  favorable  for  the  large  majority 
of  micro-organisms.     * 

The  maximum  and  minimum  temperatures  at  which 
growth  takes  place,  as  well  as  the  optimum,  are  fairly 
constant  for  each  bacterium. 

Bacteria  have  been  classified,  according  to  their 
optimum  temperature,  into— 

MIN.  OPT.  MAX. 

1.  Psychrophilic  bacteria  (chiefly  water 

organisms) o°  C  15°  C.  30°  C. 

2.  Mesophilic  bacteria  (includes  patho- 

genic bacteria) 15°  C.  27°  C.  45°  C. 

3.  Thermophilic bacteria 45°  C.  55°  C.  70°  C. 

The  thermal  death-point  of  an  organism  is  another 
biological  constant;  and  is  that  temperature  which 
causes  the  death  of  the  vegetative  forms  when  the 
exposure  is  continued  for  a  period  of  ten  minutes. 

3.  Light. — Many   organisms   are   indifferent   to   the 
presence  of  light.     On  the  other  hand,  light  frequently 
impedes  growth,  and  alters  to  a  greater  or  lesser  ex- 
tent the  biochemical  characters  of  the  organisms — 
e.  g.,  chromogenicity  or  power  of  liquefaction.     Patho- 
genic bacteria  undergo  a  progressive  loss  of  virulence 
when  cultivated  in  the  presence  of  light. 

4.  Movements. — Movements,  if  slight  and  simply  of 
a  flowing  character,  do  not  appear  to  injuriously  affect 
the  growth  of  bacteria;  but  violent  agitation,  such  as 
shaking,  absolutely  kills  them. 

A  condition  of  perfect  rest  would  seem  to  be  that 
most  conducive  to  bacterial  growth. 

The  Metabolic  Products  of  Bacteria. — Pigment  Pro- 
duction.— Many  micro-organisms  produce  one  or  more 
vivid  pigments — yellow,  orange,  red,  violet,  fluorescent, 
etc. — during  the  course  of  their  life  and  growth.  The 
colouring  matter  usually  exists  as  an  intercellular 


THE    METABOLIC    PRODUCTS    OF    BACTERIA.        123 

excrementitious  substance.  Occasionally,  however,  it 
appears  to  be  stored  actually  within  the  bodies  of  the 
bacteria.  The  chromogenic  bacteria  are  therefore 
classified,  in  accordance  with  the  final  destination  of 
the  colouring  matter  they  elaborate,  into — 

Chromoparous  Bacteria:  in  which  the  pigment  is 
diffused  out  upon  and  into  the  surrounding  medium. 

Chromophorous  Bacteria:  in  which  the  pigment  is 
stored  in  the  cell  protoplasm  of  the  organism. 

Parachromophorous  Bacteria :  in*  which  the  pigment 
is  stored  in  the  cell  wall  of  the  organism. 

Different  species  of  chromogenic  bacteria  differ  in 
their  requirements  as  to  environment,  for  the  produc- 
tion of  their  characteristic  pigments;  e.  g.,  some  need 
oxygen,  light,  or  high  temperature;  others  again  favor 
the  converse  of  these  conditions. 

Light  Production. — Some  bacteria,  and  usually  those 
originally  derived  from  water,  whether  fresh  or  salt, 
exhibit  marked  phosphorescence  when  cultivated 
under  suitable  conditions.  These  are  classed  as  "  pho- 
togenic." 

Enzyme  Production. — Many  bacteria  produce  soluble 
ferments  or  enzymes  during  the  course  of  their  growth, 
as  evidenced  by  the  liquefaction  of  gelatine,  the  clot- 
ting of  milk,  etc.  These  ferments  may  belong  to  either 
of  the  following  well-recognised  classes:  proteolytic, 
diastatic,  invertin,  rennet. 

Toxin  Production. — A  large  number,  especially  of 
the  pathogenic  bacteria,  elaborate  or  secrete  poisonous 
substances  concerning  which  but  little  exact  knowledge 
is  available,  although  many  would  appear  to  be  en- 
zymic  in  their  action. 

These  toxins  are  usually  differentiated  into — 

Extracellular  (or  Soluble)  Toxins:  those  which  are 
diffused  into,  and  held  in  solution  by,  the  surrounding 
medium. 

Intracellular  (or  Insoluble)  Toxins :  those  which  are 


124  SCHIZOMYCETKS. 

so  closely  bound  up  with  the  cell  protoplasm  of  the 
bacteria  elaborating  them  that  up  to  the  present  time 
no  means  has  been  devised  for  their  separation  or  ex- 
traction. 

End-products  of  Metabolism. — Under  this  heading 
are  included — 

Organic  Acids  (e.  g.,  lactic,  butyric,  etc.). 

Alkalies  (e.  g.,  ammonia). 

Aromatic  Compounds  (e.  g.,  indol,  phenol). 

Reducing  Substances  (e.  g.,  those  reducing  nitrates 
to  nitrites). 

Gases  (e.  g.,  sulphuretted  hydrogen,  carbon  dioxide, 
etc.). 

And  while  the  discussion  of  their  formation,  etc., 
is  beyond  the  scope  of  a  laboratory  handbook,  the 
methods  in  use  for  their  detection  and  separation 
come  into  the  ordinary  routine  work  and  will  therefore 
be  described  (vide  page  221  et  seq.). 


IX.  NUTRIENT  MEDIA. 

IN  order  that  the  life  and  growth  of  bacteria  may 
be  accurately  observed  in  the  laboratory,  it  is  neces- 
sary— 

1.  To  isolate  individual  members  of  the  different 
varieties  of  micro-organisms. 

2.  To  cultivate  isolated  organisms  apart  from  other 
associated  or  contaminating  bacteria — i.   e.,   in  pure 
culture. 

For  the  successful  achievement  of  these  objects  it  is 
necessary  to  provide  nutriment  in  a  form  suited  to  the 
needs  of  the  particular  bacterium  or  bacteria  under 
observation,  and  in  a  general  way  it  may  be  said  that 
the  nutrient  materials  should  approximate  as  closely 
as  possible,  in  composition  and  character,  to  the  natural 
soil  of  the  organism. 

The  general  requirements  of  bacteria  as  to  their 
food-supply  have  already  been  indicated  (page  121) 
and  many  combinations  of  proteids  and  of  carbohy- 
drates have  been  devised,  from  time  to  time,  on  these 
lines.  These,  together  with  various  vegetable  tissues, 
physiological  or  pathological  fluid  secretions,  etc.,  are 
collectively  spoken  of  as  nutrient  media  or  culture  media. 

The  greater  number  of  these  media  are  primarily 
fluid,  but,  on  account  of  the  rapidity  with  which  bac- 
terial growth  diffuses  itself  through  a  liquid,  it  is  im- 
possible to  study  therein  the  characteristics  of  indi- 
vidual organisms.  Many  such  media  are,  therefore, 
subsequently  rendered  solid  by  the  addition  of  sub- 
stances like  gelatine  or  agar,  in  varying  proportions, 
the  proportions  of  such  added  material  being  generally 
mentioned  when  referring  to  the  media;  e.  g.,  10  per 
cent,  gelatine,  2  per  cent.  agar.  Gelatine  is  employed 

125 


126  NUTRIENT   MEDIA. 

for  the  solidification  of  those  media  it  is  intended  to 
use  in  the  cultivation  of  bacteria  at  the  room  tem- 
perature or  in  the  "cold"  incubator.  In  the  percent- 
ages usually  employed,  gelatine  media  become  fluid  at 
25°  C. ;  higher  percentages  remain  solid  at  somewhat 
higher  temperatures,  but  the  difficulty  of  filtering 
strong  solutions  of  gelatine  militates  against  their  gen- 
eral use. 

Agar  media,  on  the  other  hand  (even  in  2  per  cent, 
solutions),  only  become  liquid  when  exposed  to  90°  C. 
for  a  considerable  period,  and  again  solidify  at  40°  C. 

When  it  becomes  necessary  to  render  these  media 
fluid,  heat  is  applied,  upon  the  withdrawal  of  which  they 
again  assume  their  solid  condition.  Such  media  should 
be  referred  to  as  liquefiable  media;  in  point  of  fact, 
however,  they  are  usually  grouped  together  with  the 
solid  media. 

NOTE. — It  must  here  be  stated  that  the  designation 
10  per  cent,  gelatine  or  2  per  cent,  agar  refers  only  to 
the  quantity  of  those  substances  actually  added  in 
the  process  of  manufacture,  and  not  to  the  percentage 
of  gelatine  or  agar,  as  the  case  may  be,  present  in 
the  finished  medium;  the  explanation  being  that  the 
commercial  products  employed  contain  a  large  propor- 
tion of  insoluble  material  which  is  separated  off  by 
filtration  during  the  preparation  of  the  liquefiable 
media. 

Other  media,  again, — e.  g.,  potato,  coagulated  blood- 
serum,  etc., — cannot  be  again  liquefied  by  physical 
means,  and  these  are  spoken  of  as  solid  media. 

The  following  pages  detail  the  method  of  preparing 
the  various  nutrient  media,  those  in  general  use  being 
printed  in  bolder  type  than  those  occasionally  required 
for  more  highly  specialised  work.  It  must  be  premised 
that  scrupulous  cleanliness  is  to  be  observed  with 
regard  to  all  apparatus,  vessels,  funnels,  etc.,  employed 
in  the  preparation  of  media. 


MEAT    EXTRACT.  12  J 

MEAT  EXTRACT. 

A  watery  solution  of  the  extractives,  etc.,  of  lean 
meat  (usually  beef)  forms  the  basis  of  several  nutrient 
media.  This  solution  is  termed  "meat  extract,"  and 
is  prepared  as  follows: 

1.  Measure  1000  c.c.  of  distilled  water  into  a  large 
flask  (or  glass  beaker,  or  enamelled  iron  pot)  and  add 
500  grammes  (roughly,  ij  pounds)  of  fresh  lean  meat, 
— e.  g.,  beefsteak  or  bullock's  heart, — finely  minced  in 
a  mincing  machine. 

2.  Heat  the  mixture  gently  in  a  water-bath,  taking 
care  that  the  temperature  of  the  contents  of  the  flask 
does  not  exceed  40°  C.  for  the  first  twenty  minutes. 
(This  dissolves  out  the  soluble  proteids,  extractives, 
salts,  etc.) 

3.  Now  raise  the  temperature  of  the  mixture  to  the 
boiling-point,   and  maintain  at  this  temperature  for 
ten  minutes.      (This  precipitates  some  of  the  albumins, 
the  haemoglobin,  etc.,  from  the  solution.) 

4.  Strain  the  mixture  through  sterile  butter  muslin 
or  a  perforated  porcelain  funnel,  then  filter  the  liquid 
through  Swedish  filter  paper  into  a  sterile  "  normal" 
litre  flask,  and  when  cold  make  up  to   1000  c.c.  by 
the  addition  of  distilled  water — to  replace  the  loss 
from  evaporation. 

5.  If  not  needed  at  once,  sterilise  the  meat  extract  in 
bulk  in  the  steam  steriliser  for  twenty  minutes  on  each 
of  three  consecutive  days. 

Calf,  sheep,  or  chicken  flesh  is  occasionally  substi- 
tuted for  the  beef;  or  the  meat  extract  may  be  pre- 
pared from  animal  viscera,  such  as  brain,  spleen,  liver, 
or  kidneys. 

NOTE. — As  an  alternative  method,  3  grammes  of 
Wyeth's  beef  juice,  invalid  bovril,  or  Liebig's  extract 
of  meat  may  be  dissolved  in  1000  c.c.  distilled  water, 
and  heated  and  filtered  as  above. 


128  NUTRIENT    MEDIA. 

Media  prepared  from  such  meat  extracts  are,  how- 
ever, eminently  unsatisfactory  when  used  for  the  cul- 
tivation of  the  more  highly  parasitic  bacteria. 

Reaction  of  Meat  Extract. — Meat  extract  thus  pre- 
pared is  acid  in  its  reaction,  owing  to  the  presence  of 
acid  phosphates  of  potassium  and  sodium,  weak  acids 
of  the  gly colic  series,  and  organic  compounds  in  which 
the  acid  character  predominates.  Owing  to  the  nature 
of  the  substances  from  which  it  derives  its  reaction, 
the  total  acidity  of  meat  extract  can  only  be  estimated 
accurately  when  the  solution  is  at  the  boiling-point. 
Moreover,  it  has  been  observed  that  prolonged  boiling 
(such  as  is  involved  in  the  preparation  of  nutrient 
media)  causes  it  to  undergo  hydrolytic  changes  which 
increase  its  acidity,  and  the  meat  extract  only  becomes 
stable  in  this  respect  after  it  has  been  maintained  at 
the  boiling-point  for  forty-five  minutes. 

Although  meat  extract  always  reacts  acid  to  phenol- 
phthalein,  it  occasionally  reacts  neutral  or  even  alka- 
line to  litmus;  and  again,  meat  extract  that  has  been 
rendered  exactly  neutral  to  litmus  still  reacts  acid  to 
phenolphthalein.  This  peculiar  behaviour  depends 
upon  two  factors: 

1 .  Litmus  is  insensitive  to  many  weak  organic  acids 
the  presence  of  which  is  readily  indicated  by  phenol- 
phthalein. 

2.  Dibasic  sodium  phosphate  which  is  formed  during 
the  process  of  neutralisation  is  a  salt  which  reacts 
alkaline  to   litmus,  but   neutral   to   phenolphthalein. 
In  order,  therefore,  to  obtain  an  accurate  estimation 
of  the  reaction  of  any  given  sample  of  meat  extract, 
it  is  essential  that — 

1 .  The  meat  extract  be  previously  exposed  to  a  tem- 
perature of  1 00°  C.  for  forty-five  minutes. 

2.  The  estimation  be  performed  at  the  boiling-point. 

3.  Phenolphthalein  be  used  as  the  indicator. 

The  estimation  is  carried  out  by  means  of  titration 


METHOD   OF   ESTIMATING  THE   REACTION.         129 


experiments  against  standard  solutions  of  caustic  soda, 
in  the  following  manner: 

Method  of  Estimating  the  Reaction. — 


Apparatus  Required : 

1.  25    c.c.    burette    graduated    in 
tenths  of  a  centimetre. 

2.  I     c.c.     pipette     graduated    in 
hundredths,   and  provided  with 
rubber    tube,    pinch-cock,    and 
delivery  nozzle. 

3.  25    c.c.    measure    (cylinder    or 
pipette,  calibrated  for  98°  C. — 
not  15°  C.). 

4.  Several  60  c.c.  conical  beakers 
or  Erlenmeyer  flasks. 

5.  White     porcelain     evaporating 
basin,  filled  with  boiling  water 
and  arranged  over  a  gas  flame 
as  a  water-bath. 

6.  Bohemian  glass  flask,  fitted  as 
a  wash-bottle,  and   filled   with 
distilled   water,  which    is   kept 
boiling  on  a  tripod  stand. 


Solutions  Required : 

1.  loN    NaOH,    accurately    stan- 
dardised. 

2.  —  NaOH,   accurately  standard- 
ised. 

3.  —  NaOH,   accurately  standard- 
ised. 

4.  0.5  per  cent,  solution  of  phenol  - 
phthalein  in  50  per  cent,  alcohol. 


METHOD. — Arrange  the  apparatus  as  indicated  in 
figure  79. 


Fig-  79- — Arrangement  of  apparatus  for  titrating. 

(A)   i.  Fill  the  burette  with  -£-  NaOH. 

2.  Fill  the  pipette  with  -^  NaOH. 

3.  Measure  25  c.c.  of  the  meat  extract  (previously 
heated  in  the  steamer  at  100°  C.  for  forty-five  minutes) 

9 


130  NUTRIENT    MEDIA. 

into  one  of  the  beakers  by  means  of  the  measure ;  rinse 
out  the  measure  with  a  very  small  quantity  of  boiling 
distilled  water  from  the  wash-bottle,  and  then  add 
this  rinse  water  to  the  meat  extract  already  in  the 
beaker. 

4.  Run  in  about  0.5  c.c.  of  the  phenolphthalein  solu- 
tion and  immerse  the  beaker  in  the  water-bath,  and 
raise  to  the  boil. 

5.  To  the  medium  in  the  beaker  run  in  -  -  NaOH 
cautiously  from   the  burette   until   the   end-point  is 


a  b  c 

Fig.  80. — a,  Sample  of  filtered  meat  extract  or  nutrient  gelatine  to  which 
phenolphthalein  has  been  added.  The  medium  is  acid,  as  evidenced  by  the 
unaltered  colour  of  the  sample,  b,  The  same  neutralised  by  the  addition  of 

--  NaOH.  The  production  of  this  faint  rose-pink  colour  indicates  that  the 
"  end-point,"  or  neutral  point  to  phenolphthalein,  has  been  reached.  If  such 
a  sample  is  cooled  down  to  say  30°  or  20°  C.,  the  colour  will  be  found  to  be- 
come more  distinct  and  decidedly  deeper  and  brighter,  resembling  that  shown 
in  c.  c,  Also  if,  after  the  end-point  is  reached,  a  further  0.5  c.c.  or  i.o  c.c. 
—  NaOH  be  added  to  the  sample,  the  marked  alkalinity  is  evidenced  by  the 
deep  colour  here  shown. 

reached,  as  indicated  by  the  development  of  a  pinkish 
tinge,  shown  in  figure  80.  Note  the  amount  of  deci- 
normal  soda  solution  used  in  the  process. 

NOTE. — Just  before  the  end-point  is  reached,  a  very 
slight  opalescence  may  be  noted  in  the  fluid,  due  to 
the  precipitation  of  dibasic  phosphates.  After  the 
true  end-point  is  reached,  the  further  addition  of  about 
0.5  c.c.  of  the  decinormal  soda  solution  will  produce 
a  deep  magenta  colour  (Fig.  80,  c),  which  is  the  so-called 
"end-point"  of  the  American  Committee  of  Bacteri- 
ologists. 


METHOD    OF    EXPRESSING   THE    REACTION.         131 

(B)  Perform  a  "control"  titration  (occasionally  two 
controls  may  be  necessary),  as  follows: 

1.  Measure  25  c.c.  of  the  meat  extract  into  one  of  the 
beakers,   wash  out  the  measure  with  boiling  water, 
and  add  the  phenolphthalein  as  in  the  first  estimation. 

2.  Run  in  -y-  NaOH  from  the  pipette,  just  short  of 
the  amount  required  to  neutralise  the  25  c.c.  of  medium. 
(For  example,  if  in  the  first  estimation  5  c.c.  of  -—• 
NaOH  were  required  to  render   25   c.c.   of  medium 
neutral  to  phenolphthalein,  only  add  0.48  c.c.  of  -j- 
NaOH.)     Immerse  the  beaker  in  the  water-bath. 

3.  Complete  the  titration  by  the  aid  of  the  •—  NaOH. 

4.  Note  the  amount  of  -£-  NaOH  solution  required 
to  complete  the  titration,  and  add  it  to  the  equivalent 
of  the  -y-  NaOH  solution  previously  run  in.     Take 
the  total  as  the  correct  estimation. 

Method  of  Expressing  the  Reaction.— 

The  reaction  or  litre  of  meat  extract,  medium, 
or  any  solution  estimated  in  the  foregoing  manner,  is 
most  conveniently  expressed  by  indicating  the  number 
of  cubic  centimetres  of  normal  alkali  (or  normal  acid) 
that  would  be  required  to  render  one  litre  of  the  solu- 
tion exactly  neutral  to  phenolphthalein. 

The  sign  +  (plus)  is  prefixed  to  this  number  if  the 
solution  reacts  acid,  and  the  sign  —  (minus)  if  it  reacts 
alkaline. 

For  example,  "meat  extract  +  10,"  indicates  a 
sample  of  meat  extract  which  reacts  acid  to  phenol- 
phthalein, and  would  require  the  addition  of  10  c.c. 
of  normal  NaOH  per  litre,  to  neutralise  it. 

NOTE. — Such  a  solution  would  probably  react  alka- 
line to  litmus. 

Conversely,  if  as  the  result  of  our  titration  experi- 
ments we  find  that  25  c.c.  of  meat  extract  require  the 
addition  of  5  c.c.  -~  NaOH  to  neutralise,  then  1000  c.c. 


132 


NUTRIENT   MEDIA. 


of  meat  extract  will  require  the  addition  of  200  c.c.  -^- 

NaOH  ==  20  c.c.  -f  NaOH. 

And  this  last  figure,  20,  preceded  by  the  sign  -f- ,  to 

signify  that  it  is  acid, 
indicates  the  reaction  of 
the  meat  extract. 

NOTE. — The  standard 
soda  solutions  should  be 
prepared  by  accurate 
measuring  operations, 
controlled  by  titrations, 
from  a  stock  solution 
of  loN  NaOH,  which 
should  be  very  care- 
fully standardised.  This 
stock  solution  must  be 
kept  in  an  aspirator  bot- 
tle to  which  air  can  only 
gain  access  after  it  has 
been  dried  and  rendered 
free  from  CO2.  This  may 
be  done  by  first  leading 
it  over  H2SO4  and  soda 
lime,  or  soda  lime  alone, 
by  some  such  arrange- 
ment  as  is  shown  in 
figure  8 1,  which  also 
Fig.  81.— Soda  bottle.  shows  a  constant  burette 

arrangement  for  the  de- 
livery of  small  measured  quantities  of  the  dekanormal 
soda  solution. 


STANDARDISATION  OF  MEDIA. 

Differences  in  the  reaction  of  the  medium  in  which 
it  is  grown  will  provoke  not  only  differences  in  the  rate 
of  growth  of  any  given  bacterium,  but  also  well-marked 
differences  in  its  cultural  and  morphological  characters ; 
and  nearly  every  organism  will  be  found  to  affect  a 
definite  "  optimum  reaction" — a  point  to  be  carefully 
determined  for  each .  Fortunately,  however,  the  ' '  opti- 


STANDARDISING    NUTRIENT    BOUII^ON.  133 

mum"  usually  approximates  fairly  closely  to  -f-io; 
and  as  experiment  has  shown  that  this  reaction  is  the 
most  generally  useful  for  laboratory  work,  it  is  the 
one  which  may  be  adopted  as  the  standard  for  all  nu- 
trient media  derived  from  meat  extract. 

Briefly,  the  method  of  standardising  a  litre  of  media 
to  +10  consists  in  subtracting  10  from  the  initial  litre 
of  the  medium  mass;  the  remainder  indicates  the 
number  of  cubic  centimetres  of  normal  soda  solution 
that  must  be  added  to  the  medium,  per  litre,  to  render 
the  reaction  -fio. 

Standardising  Nutrient  Bouillon. — For  example,  1000 
c.c.  bouillon  are  prepared;  on  titration  it  is  found 

1.  25  c.c.  require  the  addition  of  5.50  c.c.  -£-  NaOH 
to  neutralise. 

2.  25  c.c.  require  the  addition  of  5.70  c.c.  -~  NaOH 
to  neutralise. 

3.  25  c.c.  require  the  addition  of  5.60  c.c.  -~  NaOH 
to  neutralise. 

2  and  3  are  controls.  Averaging  these  two  controls, 
25  c.c.  require  the  addition  of  5.65  c.c.  -—-  NaOH  to 
neutralise,  and  therefore  1000  c.c.  require  the  addition 
of  226  c.c.  -£-  NaOH,  or  22.60  -f  NaOH. 

Initial  titre  of  the  bouillon  =  +22.6,  and  as  such 
requires  the  addition  of  (22.6  c.c.  —  10  c.c.)  =  12.6  c.c. 
of -7-  NaOH  per  litre  to  leave  its  finished  reaction  + 10. 

But  the  three  titrations,  each  on  25  c.c.  of  medium, 
have  reduced  the  original  bulk  of  the  bouillon  to  (1000 
—  75  c.c.)  =  925  c.c.  The  amount  of  -^-  NaOH  re- 
quired to  render  the  reaction  of  this  quantity  of  medium 
H-IO  may  be  deduced  thus: 

1000  c.c.  :  925  c.c.  ::  12.6  c.c.  :  x. 

Then  x  ==  11.65  c.c.  -f-  NaOH. 

Whenever  possible,  however,  the  required  reaction 
is  produced  by  the  addition  of  dekanormal  soda  solu- 
tion, on  account  of  the  minute  increase  it  causes  in  the 


134 


NUTRIENT   MEDIA. 


bulk,  and  the  consequent  insignificant  disturbance  of 
the  percentage  composition  of  the  medium.  By  means 
of  a  pipette  graduated  to  o.oi  c.c.  it  is  possible  to  de- 
liver very  small  quantities;  but  if  the  calculated  amount 
runs  into  thousandth  parts  of  a  cubic  centimetre,  these 
are  replaced  by  corresponding  quantities  of  normal  soda. 

In  the  above  example  it  is  necessary  to  add  11.65 
c.c.  normal  NaOH  or  its  equivalent,  1.165  c-c-  deka- 
normal  NaOH.  The  first  being  too  bulky  a  quantity, 
and  the  second  inconveniently  small  for  exact  measure- 
ment, the  total  weight  of  soda  is  obtained  by  substi- 
tuting 1. 1 6  c.c.  dekanormal  soda  solution,  and  0.05 
c.c.  of  normal  soda  solution. 

Standardising  Nutrient  Gelatine. — In  the  finished 
medium  it  has  been  found  experimentally  that  every 
cubic  centimetre  of  meat  extract  has  increased  in  bulk 
to  1.008  c.c.  for  every  o.oi  gramme  of  added  gelatine, 
and  this  factor  must  be  taken  into  account  in  calcu- 
lating the  amount  of  soda  solution  necessary  to  pro- 
duce the  standard  reaction  in  the  finished  medium. 

The  following  expansion  table  shows  at  a  glance  the 
increase  in  bulk  for  the  varying  percentages: 

GELATINE  EXPANSION  TABLE. 

AT  20°  CENTIGRADE. 
loo  c.c.  meat  extract 
+  5  gms.  gelatine  measure 
+  6 
+  7 
+  8 
+  9 
+  10 


1000  c.c.  meat  extract 

asure  iO4.oc 

+  5°gms-  gelatine  measure  1040  c  c 

104.8 

+  60 

1048 

105.6 

+   70 

1056 

106.4 

+  80 

1064 

107.2 

+  9Q 

1072 

108.0 

+  100 

I080 

108.8 

+  110 

1088 

109.6 

+  I2O 

1096 

110.4 

+  130 

1104 

III.  2 

+  140 

III2 

II2.0 

+  150 

1120 

116.0 

+  2OO 

1160 

+  12 
+  13 

+  H 

+15 

+  20 

The  method  of  standardising  gelatine  is  very  similar 
to  that  described  under  bouillon. 


STANDARDISING    NUTRIENT   AGAR.  135 

For  example,  1000  c.c.  of  meat  extract  are  employed 
in  the  manufacture  of  1 1  per  cent,  gelatine. 

By  referring  to  the  above  table  it  is  found  that  after 
solution  the  medium  mass  measures  1088  c.c.,  and  if 
three  titrations  are  performed  there  still  remain  1013 
c.c.  of  medium  to  be  standardised  (instead  of  only 
925  c.c.). 

If  the  initial  reaction  of  the  gelatine  mass  is  +  24,  it 
will  require  the  addition  of  14  c.c.  -7-  NaOH  per  litre 
to  render  the  reaction  of  the  finished  medium  + 10. 

Therefore,  1000  c.c.  :  1013  c.c.  : :  14  c.c.  :  x. 

x==  14.182  c.c.  -^-NaOH. 

This  quantity  is  replaced  by 

1.41  c.c.  dekanormal  soda  solution  (equivalent  to  14.100  —  NaOH)     . 
0.08     "    normal  soda  solution          (          "  "    0.080  y  NaOH) 

0.02     "    decinormal  soda  solution    (          "  "    0.002  —  NaOH) 

14.182 

giving  the  requisite  weight  of  soda  in  a  little  over  1.5 
c.c.  of  fluid. 

Standardising  Nutrient  Agar. — Every  cubic  centi- 
metre of  meat  extract  increases  in  bulk  to  1.0053  c-c- 
for  every  o.oi  gramme  of  added  agar. 

The  following  table  gives  the  calculated  bulk  for 
varying  percentage  of  agar: 


AGAR  EXPANSION  TABLE. 

AT  20°  CENTIGRADE. 

100  c.c.  meat  extract  1000  c.c.  meat  extract 

-(-0.5  gm.  agar  measures  100.265  C.c.  -f-   5    agar  measures   1002.65  c>c- 

-f  i.o    "     "  "         100.530  "  +10     "  "          1005.30    " 

-f-i.5gms.  "  "         100.795   "  +  15      "  "          1007.95    " 

-j-2.0    "     "  "          101.060  "  -(-20      "  "          1010.60    " 

The  method  of  standardising  nutrient  agar  is  similar 
to  that  described  under  nutrient  gelatine,  and  a  refer- 
ence to  the  example  there  given  will  at  once  explain 
the  necessary  steps  in  the  calculation. 


136  NUTRIENT   MEDIA. 

THE  FILTRATION  OF  MEDIA. 

Fluid  media  are  usually  filtered  through  stout  Swed- 
ish filter  paper  (occasionally  through  a  porcelain  filter 
candle),  and  in  order  to  accelerate  the  rate  of  filtration 
the  filter  paper  should  be  folded  in  that  form  which 
is  known  as  the  "physiological  filter,"  not  in  the  ordi- 
nary "  quadrant"  shape,  as  by  this  means  a  large  surface 
is  available  for  filtration  and  a  smaller  area  in  contact 
with  the  glass  funnel  supporting  it. 

To  fold  the  filter  proceed  thus: 

1.  Take  a  circular  piece  of  filter  paper  and  fold  it 
exactly  through  its  centre  to  form  a  semicircle. 

2.  Fold  the  semicircle  exactly  in  half  to  form  a  quad- 
rant; make  the  crease  distinct  by  running  the  thumb- 
nail along  it,  then  open  the  filter  out  to  a  semicircle 
again. 

3.  Fold  each  end  of  the  semicircle  in  to  the  centre 
and  so  form  another  quadrant;  smooth  down  the  two 
new  creases  thus  formed  and  again  open  out  to  a  semi- 
circle. 

4.  The  semicircle  now  appears  as  in  figure  82,  a,  the 
dark  lines  indicating  the  creases  already  formed. 

5.  Fold  the  point  i  over  to  the  point  3,  and  ia  to 
3a,  to  form  the  creases  4  and  4a,  indicated  in  the  dia- 
gram by  the  light  lines.     Fold  point  i  over  to  3,  and 
i  a  to  3a,  to  form  the  creases  5  and  5*1. 

6.  Thus  far  the  creases  have  all  been  made  on  the 
same  side  of  the  paper.     Now  subdivide  each  of  the 
eight  sectors  by  a  crease  through  its  centre  on  the 
opposite  side  of  the  paper,  indicated  by  the  broken  line 
in  the  diagram.     Fold  up  the  filter  gradually  as  each 
crease  is  made,  and  when  finished  the  filter  has  assumed 
the  shape  of  a  wedge,  as  in  figure  82,  b. 

When  opened  out  the  filter  assumes  the  shape  repre- 
sented in  figure  82,  c. 

The  folded  filter  is  next  placed  inside  a  glass  funnel 


FILTRATION    OF    SOLID    MEDIA. 


137 


supported  on  a  retort  stand  and  moistened  with  hot 
distilled  water  before  the  filtration  of  the  medium  is 
commenced. 

Solid  media  are  filtered  through  a  specially  made 
filter  paper, — "  papier  Chardin," — which  is  sold  in 
boxes  of  twenty-five  ready-folded  filters. 


Fig.   82. — Filter   folding :    <?,   Filter  folded  in    half,   showing  creases  ;    />, 
appearance  of  filter  on  completion  of  folding ;  c,  filter  opened  out  ready  for 


Gelatine,  when  properly  made,  filters  through  this 
paper  as  quickly  as  bouillon  does  through  the  Swedish 
filter  paper,  and  does  not  require  the  use  of  the  hot- 
water  funnel. 

Agar,  likewise,  if  properly  made,  filters  readily, 
although  not  at  so  rapid  a  rate  as  gelatine.  If  badly 


138 


NUTRIENT   MEDIA. 


"egged,"  and    also    during   the  winter  months,  it  is 
necessary  to  surround  the  glass  funnel,  in  which  the 

filtration  of  the  agar  is 
carried  on,  by  a  hot- 
water  jacket.  This  is 
done  by  placing  the  glass 
funnel  inside  a  double- 
walled  copper  funnel — 
the  space  between  the 
walls  being  filled  with 
water  at  about  90°  C. — 
and  supporting  the  lat- 
ter on  a  ring  gas  burner 
fixed  to  a  retort  stand 
(Fig.  83).  The  gas  is 
lighted  and  the  water 
jacket  maintained  at  a 
high  temperature  until 
filtration  is  completed. 


Fig.  83. — Ring  burner  and  hot-water 
filter. 


TUBING  NUTRIENT  MEDIA. 

After  the  final  filtration,  the  nutrient  medium  is 
"  tubed" — i.  e.,  filled  into  sterile  tubes  in  definite  meas- 
ured quantities,  usually  10  c.c.;  or  "flasked" — i.  e., 
filled  into  sterile  flasks  in  fairly  large  quantities.  This 
process  is  sometimes  carried  out  by  means  of  a  large 
separator  funnel  fitted  with  a  "three-way"  tap  which 
communicates  with  a  small  graduated  tube  (capacity 
20  c.c.  and  graduated  in  cubic  centimetres)  attached  to 
the  side.  The  shape  of  this  piece  of  apparatus,  known 
as  Treskow's  funnel,  renders  it  particularly  liable  to 
damage.  It  is  better,  therefore,  to  arrange  a  less  ex- 
pensive piece  of  apparatus  which  will  serve  the  pur- 
pose equally  well  (Fig.  84). 

A  Geissler's  three-way  stop-cock  has  the  tube  on 
one  side  of  the  tap  ground  obliquely  at  its  extremity, 


TUBING    NUTRIENT   MEDIA. 


139 


the  tube  on  the  opposite  side  cut  off  within  3  c.c.  of  the 
tap.  The  short  tube  is  connected  by  means  of  a  per- 
forated rubber  cork  with  a  10  c.c.  length  of  stout  glass 
tubing  (1.5  c.c.  bore).  The  third  channel  of  the  three- 
way  tap  is  connected,  by  means  of  rubber  tubing,  with 
the  nozzle  of  an  ordinary  separator  funnel.  Finally, 
the  receiving  cylinder  above  the  three-way  tap  is  gradu- 
ated in  cubic  centimetres  up  to  20,  by  pouring  into  it 


Fig.  84. — Three-way  tap,  home-made. 


Fig.  85.— Gas  tube 
(Durham). 


measured  quantities  of  water  and  marking  the  various 
levels  on  the  outside  with  a  writing  diamond. 

Fluid  media  containing  carbohydrates  are  filled  into 
fermentation  tubes  (vide  Fig.  14);  or  into  ordinary 
media  tubes  which  already  have  smaller  tubes,  inverted, 
inside  them  (Fig.  85),  to  collect  the  products  of  growth 
of  gas-forming  bacteria.  When  first  filled,  the  small 
tubes  float  on  the  surface  of  the  medium ;  after  the  first 
sterilisation  nearly  all  the  air  is  replaced  by  the  medium, 


140  NUTRIENT   MEDIA. 

and  after  the  final  sterilisation  the  gas  tubes  will  be 
submerged  and  completely  filled  with  the  medium. 

Storing  "Tubed"  Media. — Media  after  being  tubed 
are  best  stored  by  packing,  in  the  vertical  position, 
in  oblong  boxes  having  an  internal  measurement  of 
37  cm.  long  by  12  cm.  wide  by  10  cm.  deep.  Bach 
box  (Fig.  86)  has  a  movable  partition  formed  by  the 
vertical  face  of  a  weighted  triangular  block  of  wood, 
sliding  free  on  the  bottom  (Fig.  86,  A);  or  by  a  flat 


Fig.  86. — Medium  box,  showing  alternative  partitions  A  and  B. 

piece  of  wood  sliding  in  a  metal  groove  in  the  bottom 
of  the  box,  which  can  be  fixed  at  any  spot  by  tightening 
the  thumbscrew  of  a  brass  guide  rod  which  transfixes 
the  partition  (Fig.  86,  B).  The  front  of  the  box  is  pro- 
vided with  a  handle  and  a  celluloid  label  for  the  name 
of  the  contained  medium.  These  boxes  are  arranged 
upon  shelves  in  a  dark  cupboard, — or  preferably  an  iron 
safe, — which  should  be  rendered  as  nearly  air-tight  as 
possible,  and  should  have  the  words  "media  stores" 
painted  on  its  doors. 


X.  CULTURE  MEDIA. 

Nutrient  Bouillon. — 

1.  Measure   800   c.c.  of   meat  extract   into   a  litre 
flask. 

2.  Weigh  out  Witte's  peptone,  10  grammes  (=  i  per 
cent.),  salt,  5  grammes  (=  0.5  per  cent.),  and  mix  into 
a  smooth  paste  with  200  c.c.  of  meat  extract  previously 
heated  to  60°  C.     (Be  careful  to  leave  no  unbroken 
globular  masses  of  peptone.) 

3.  Add  the  peptone  emulsion  to  the  meat  extract  in 
the  flask  and  heat  the  steamer  for  forty-five  minutes 
(to  completely  dissolve  the  peptone,  and  to  render  the 
acidity  of  the  meat  extract  stable). 

4.  Estimate  the  reaction  of  the  medium;  control  the 
result;   render   the   reaction   of   the   finished   medium 
+  10  (vide  page  133). 

5.  Heat  for  half  an  hour  in   the   steamer  at   100° 
C.    (to  complete  the  precipitation  of  the  phosphates, 
etc.). 

6.  Filter  through  Swedish  filter  paper  into  a  sterile 
flask. 

7.  Fill  into  sterile  tubes  (10  c.c.  in  each  tube). 

8.  Sterilise  in  the  steamer  for  twenty  minutes  on 
each  of  three  consecutive  days — i.  e.,  by  the  discon- 
tinuous method  (vide  page  41). 

Inosite-free  Media  Bouillon  (Durham).— 

1.  Prepare  meat  extract,  1000  c.c.  (vide  page  127),  from 
bullock's  heart  which  has  been  "hung"   for  a  couple  of 
days. 

2.  Prepare  nutrient  bouillon  (-J-io),  1000  c.c.  (vide  supra), 
from  the  meat  extract,  and  store  in  i -litre  flask. 

3.  Inoculate  the  bouillon  from  a  pure  cultivation  of  the 
B.  lactis  aerogenes,  and  incubate  at  37°  C.  for  forty-eight 
hours. 

141 


142  CULTURE    MEDIA. 

4.  Heat  in  the  steamer  at  100°  C.  for  twenty  minutes  to 
destroy  the  bacilli  and  some  of  their  products. 

5.  Estimate  the  reaction  of  the  medium  and  if  necessary 
restore  to  -|-io. 

6.  Inoculate  the  bouillon  from  a  pure  cultivation  of  the 
B.   coli  communis  and  incubate  at  37°  C.  for  forty-eight 
hours. 

7.  Heat  in  the  steamer  at  100°  C.  for  twenty  minutes. 
Now  fill  two  fermentation  tubes  with  the  bouillon,  tint 

with  litmus  solution,  and  sterilise;  inoculate  with  B.  lactis 
aerogenes.  If  no  acid  or  gas  is  formed,  the  bouillon  is  in 
a  sugar-free  condition;  but  if  acid  or  gas  is  present,  again 
make  the  bouillon  in  the  flask  -f-io,  reinoculate  with  one  or 
other  of  the  above-mentioned  bacteria,  and  incubate ;  then 
test  again.  Repeat  this  till  neither  acid  nor  gas  appears 
in  the  medium. 

8.  After  the  final  heating,  stand  the  flask  in  a  cool  place 
and  allow  the  growth  to  sediment.     Filter  the  supernatant 
broth  through  Swedish  filter  paper.     If  the  filtrate  is  cloudy, 
filter  through  a  porcelain  filter  candle. 

9.  Tube,  and  sterilise  as  for  bouillon. 

Bouillon  prepared  in  the  above-described  manner  will 
prove  to  be  absolutely  sugar-free;  and  from  it  may  be  pre- 
pared nutrient  sugar-free  gelatine  or  agar,  by  following  the 
directions  given  on  pages  145  and  149,  respectively,  substi- 
tuting the  inosite-free  meat  extract  for  the  ordinary  meat 
extract.  The  most  important  application  of  inosite-free 
bouillon  is  its  use  in  the  preparation  of  sugar  bouillons, 
whether  glucose,  maltose,  lactose,  or  saccharose,  of  exact 
percentage  composition. 

Glycerine  Bouillon. — 

1.  Measure  out  nutrient  bouillon,    1000  c.c.    (vide  page 
141,  sections  i  to  6). 

2.  Measure  out  glycerine,  60  c.c.  (=  6  per  cent.),  and  add 
to  the  bouillon. 

3.  Tube,  and  sterilise  as  for  bouillon. 
Sugar  Bouillon. — 

1.  Measure  out  nutrient  bouillon,    1000  c.c.    (vide  page 
141,  sections  i  to  6). 

2.  Weigh  out  glucose   (anhydrous),   20  grammes  (=   2 
per  cent.),  and  dissolve  in  the  fluid. 

3.  Tube,  and  sterilise  as  for  bouillon. 

Ordinary  commercial  glucose  serves  the  purpose  equally 
well,  but  is  not  recommended,  as  during  the  process  of 
sterilisation  the  medium  gradually  deepens  in  colour. 

NOTE. — In  certain  cases  a  corresponding  percentage 
of  lactose,  maltose,  or  saccharose  is  substituted  for  glu- 
cose. 


NUTRIENT   BOUILLON.  143 

Glucose  Formate  Bouillon  (Kitasato). — 

1.  Measure  out  nutrient  bouillon,    1000  c.c.    (vide  page 
141,  sections  i  to  6). 

2.  Weigh  out  glucose,  20 grammes  (—  2  percent.),  sodium 
formate,  4  grammes  (—  0.4  per  cent.),   and  dissolve  in  the 
fluid. 

3.  Tube,  and  sterilise  as  for  bouillon. 
Sulphindigotate  Bouillon   (Weyl).— 

1.  Measure  out  nutrient  bouillon  (vide  page  141,  sections 
i  to  6). 

2.  Weigh    out    glucose,    20   grammes    (—  2    per   cent.), 
sodium  sulphindigotate,   i  gramme  (=0.1  per  cent.),  and 
dissolve  in  the  fluid. 

3.  Tube,  and  sterilise  as  for  bouillon. 

NOTE. — The  finished  medium  is  of  a  blue  colour,  which 
during  the  growth  of  anaerobic  bacteria  is  oxidised  and 
decolourised  to  a  light  yellow. 

Nitrate  Bouillon.— 

1.  Measure  out  nutrient  bouillon,    1000  c.c.    (vide  page 
141,  sections  i  to  6). 

2.  Weigh  out  potassium  nitrate,  5  grammes  (=  0.5  per 
cent.),  and  dissolve  it  in  the  bouillon. 

3.  Tube,  and  sterilise  as  for  bouillon. 

NOTE. — The  nitrate  of  sodium  or  ammonium  may  be 
substituted  for  that  of  potassium,  or  the  salt  may  be  added 
in  the  proportion  of  from  o.  i  to  i  per  cent,  to  meet  special 
requirements. 

Iron  Bouillon. — 

1.  Measure  out  nutrient  bouillon,    1000  c.c.    (vide  page 
141,  sections  i  to  6). 

2.  Weigh  out  ferric  tartrate,  i  gramme  (—  o.i  per  cent.), 
and  dissolve  it  in  the  bouillon. 

3.  Tube,  and  sterilise  as  for  bouillon. 

N.  B. — The  lactate  of  iron  may  be  substituted  for  the 
tartrate. 

Lead  Bouillon. — 

1.  Measure  out  nutrient  bouillon,  1000  c.c.  (vide  page  141, 
sections  i  to  6). 

2.  Weigh  out  lead  acetate,  i  gramme  (—  o.i   percent.), 
and  dissolve  it  in  the  bouillon. 

3i  Tube,  and  sterilise  as  for  bouillon. 
Litmus  Bouillon. — 

1.  Measure  out  nutrient  bouillon,    1000  c.c.    (vide  page 
141,  sections  i  to  6). 

2.  Add  sufficient  sterile  litmus  solution  to  tint  the  medium 
a  dark  lavender  colour.     (Media  rendered  -f-io  will  usually 
react  very  faintly  alkaline  or  occasionally  neutral  to  litmus.) 

3.  Tube,  and  sterilise  as  for  bouillon. 


144  GUI/TURK    MEDIA. 

Lactose  Litmus  Bouillon  (Lakmus  Molke). — 

1.  Weigh  out  peptone,  4  grammes,  and  emulsify  it 
with  200  c.c.  meat  extract  (vide  page  127),  previously 
heated  to  60°  C. 

2.  Weigh  out  salt,  2  grammes,  and  lactose,  20,  and 
mix  with  the  emulsion. 

3.  Wash  the  mixture  into  a  sterile  litre  flask  with 
200  c.c.  meat  extract  and  add  600  c.c.  distilled  water. 

4.  Heat  in  the  steamer  at  100°  C.  for  thirty  minutes, 
to  completely  dissolve  the  peptone,  etc. 

5.  Neutralise  carefully  to  litmus  paper  by  the  suc- 
cessive additions  of  small  quantities  of  decinormal  soda 
solution. 

6.  Replace  in  the  steamer  for  twenty  minutes  to 
precipitate  phosphates,  etc. 

7.  Filter  through  two  thicknesses  of  Swedish  filter 
paper. 

8.  Add  sterile  litmus  solution,  sufficient  to  colour 
the  medium  a  deep  purple. 

9.  Tube,  and  sterilise  as*  for  bouillon. 


Parietti's  Bouillon.— 

1.  Measure  out  pure  hydrochloric  acid,  4  c.c.,  and  add 
to  it  carbolic  acid  solution  (5  per  cent.),   100  c.c.     Allow 
the  solution  to  stand  at  least  a  few  days  before  use. 

2.  This  solution  is  added  in  quantities  of  o.i,  0.2,  and 
0.3  c.c.  (delivered  by  means  of  a  sterile  graduated  pipette) 
to  tubes   each  containing   10  c.c.   of  previously  sterilised 
nutrient  bouillon  (vide  page  141). 

3.  Incubate  at  37°  C.  for  forty-eight  hours  to  eliminate 
contaminated  tubes.     Store  the  remainder  for  future  use. 

Carbolised  Bouillon. — 

1.  Prepare  nutrient  bouillon  (vide  page  141,  sections  i  to 
6). 

2.  Weigh  out  carbolic  acid,  i  gramme  (2.5  or  5  grammes 
may  be  needed  for  special  purposes),  and  dissolve  it  in  the 
medium. 

3.  Tube,  and  sterilise  as  for  bouillon. 


NUTRIENT  GELATINE.  145 

Nutrient  Gelatine.— 

1.  Measure  out  meat  extract,  800  c.c.,  into  a  2-litre 
flask. 

2.  Weigh  out  Witte's  peptone,  10  grammes  (=  i  per 
cent.),  salt,    5   grammes   (=0.5  per  cent.),  and   mix 
into  a  smooth  paste  with  200  c.c.  meat  extract  pre- 
viously heated  to  60°  C. ;  add  the  emulsion  to  the  meat 
extract  in  the  flask. 

.3.  Weigh  out  that  quantity  of  best  Gold  Label  French 
gelatine  necessary  to  secure  the  required  percentage,— 
usually  90  to  120  grammes  (  =  9  per  cent,  to  12  per 
cent.), — cut  the  sheets  into  small  pieces  and  add  to  the 
meat  extract. 

4.  Place  the  flask  in  the  steamer  at  100°  C.  for  one 
hour. 

5.  Estimate  the  reaction  of  the  medium  mass  and 
control  the  result;  then  add  sufficient  soda  solution  to 
render  the  reaction  of  the  calculated  bulk  of  medium 
+  10.     (See  Gelatine  Expansion  Table,  page   134.) 

6.  Replace  in  the  steamer  for  twenty  minutes  (to 
complete  the  precipitation  of  the  phosphates). 

7.  Allow  the  medium  mass  to  cool  to  60°  C.     Well 
whip  the  whites  of  two  eggs,  add  to  the  contents  of  the 
flask,  and  replace  in  the  steamer  at  100°  C.  for  about 
half  an  hour   (until  the  egg-albumen  has  coagulated 
and  formed  large,  firm  masses  floating  on  and  in  clear 
gelatine). 

8.  Filter  through  papier  Chardin  into  a  sterile  flask. 

9.  Tube  in  quantities  of  10  c.c. 

10.  Sterilise  in  the  steamer  at  100°  C.  for  twenty 
minutes  on  each  of  three  consecutive  days — i.  e.,  by 
the  discontinuous  method. 

Rapid  Method  of  Preparing  Nutrient  Gelatine. — 
i .  Finely  mince  500  grammes  of  lean  beef  and  add  to 
800  c.c.  of  distilled  water  in  a  flask;  place  the  flask 
in  a  water-bath,  and  raise  the  temperature  of  its  con- 
tents to  and  keep  at  45°  C.  for  twenty  minutes;  then 


146 


GUI/TURK  MEDIA. 


rapidly  raise  the  temperature  to  100°  C.,  and  maintain 
there  for  ten  minutes. 

2.  Weigh  a  2 -litre  flask  on  a  trip  balance  (Fig.  87) 
and  note  the  weight,  or  counterpoise  carefully.     Filter 


Fig.  87. — Trip  balance. 

the  mixture  into  the  flask.     Do  not  make  up  the  fil- 
trate to  1000  c.c.  as  in  the  ordinary  method. 

An  extremely  useful  counterpoise  is  a  small  sheet- 
brass  cylinder  about  38  mm.  high  and  38  mm.  in  di- 
ameter, with  a  funnel-shaped  top  and  provided  with  a 
side  tube  by  which  its  contents,  fine  "dust"  shot,  may 
be  emptied  out  (Fig.  88). 

3.  Weigh  out  and  mix  10  grammes  of  peptone,  5 
grammes  of  salt,  and  make  into  a 
thick  paste  with  150  c.c.  distilled 
water;  then  add  the  emulsion  to 
the  meat  extract  in  the  flask;  also 
add  100  grammes  sheet  gelatine 
cut  into  small  pieces,  and  return 
the  flask  to  the  water-bath. 

4.  Arrange  a  lo-litre  tin  can 
(with  copper  bottom,  such  as  is 
used  in  the  preparation  of  distilled 
water)  by  its  side,  fill  the  can  with 
boiling  water  and  place  a  lighted 
Bunsen  burner  under  it.  Fit  a 
long  safety  tube  to  the  neck  of  the  can  and  also  a  deliv- 
ery tube,  bent  twice  at  right  angles,  adjusted  to  reach 
to  the  bottom  of  the  interior  of  the  flask  (Fig.  89). 


u 


Fig.  88.  —  Counter- 
poise ;  weight  when  emp- 
,ty»  35  grammes;  when 
full  of  dust  shot,  200 
grammes. 


NUTRIENT   GELATINE. 


147 


5.  Keep  the  water  in  the  can  vigourously  boiling,  and 
so  steam  at   100°  C.,  bubbling  through  the  medium 
mass,  for  ten  minutes,  by  which  time  complete  solution 
of  the  gelatine  is  effected. 

6.  Weigh  the  flask  and  its  contents;  then   (1115* 
grammes  +  weight  of  the  flask)  minus  (weight  of  the 


Fig.  89. — Steam  can  arrangement  for  media. 


flask  and  its  contents)  equals  the  weight  of  water  re- 
quired to  make  up  the  bulk  to  i  litre.  Add  the  requi- 
site quantity  of  water  at  the  boiling-point. 

7.  Titrate  and  estimate  the  reaction  of  the  medium 

1  This  figure  is  obtained  by  adding  together  I  litre  water,  1000  grammes ; 
IO  per  cent,  gelatine,  100  grammes;  I  percent,  peptope,  IO  grammes;  0.5 
per  cent,  salt,  5  grammes  ;  total,  1115  grammes.  Modifications  of  the  above 
process,  as  to  quantities  and  percentages,  will  require  corresponding  alterations 
of  the  figures.  The  average  weight  of  I  litre  of  IO  per  cent,  nutrient  gela- 
tine when  prepared  in  this  way  is  1080  grammes  (compare  expansion  table, 
page  134). 


148  CULTURE  MEDIA. 

mass;  control  the  result.  Calculate  the  amount  of 
soda  solution  required  to  make  the  reaction  of  the 
medium  mass  +10  (i.  e.,  calculate  for  1000  c.c.,  less 
the  quantity  used  for  the  titrations). 

8.  Add  the  necessary  amount  cf  soda  solution  and 
heat  in  the  steamer  at  100°  C.  for  twenty  minutes,  to 
precipitate  the  phosphates,  etc. 

9.  Clarify  with  egg;  filter,  tube,  and  sterilise  as  for 
nutrient  gelatine  (vide  page  145). 

Sugar  Gelatine. — 

1.  Prepare  nutrient  gelatine  (vide  page  145,  sections  i  to 

7). 

2.  Weigh  out  glucose,  20  grammes  (==  2  per  cent.),  and 
dissolve  in  the  hot  gelatine. 

3.  Filter  through  papier  Chardin. 

4.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

NOTE. — In  certain  cases,  lactose,  maltose,  or  saccharose, 
in  similar  percentage,  is  substituted  for  glucose. 
Glucose  Formate  Gelatine  (Kitasato). — 

1.  Prepare  nutrient  gelatine  (Me  page  145,  sections  i  to 

7). 

2.  Weigh  out  glucose,  20  grammes  (=  2  per  cent.),  and 
sodium  formate,  4  grammes  (=  0.4  per  cent.),  and  dissolve 
in  the  hot  gelatine. 

3.  Filter  through  papier  Chardin. 

4.  Tube,  and  sterilise  as  for  nutrient  gelatine. 
Sulphindigotate  Gelatine  (Weyl).— 

1.  Prepare  nutrient  gelatine   (vide  page   145,   sections   i 

to  7). 

2.  Weigh  out  glucose,  20  grammes  (=  2  per  cent.),  and 
sodium  Sulphindigotate,   i  gramme  (=0.1  per  cent.),  and 
dissolve  in  the  hot  gelatine. 

3.  Filter  through  papier  Chardin. 

4.  Tube,  and  sterilise  as  for  nutrient  gelatine. 
Litmus  Gelatine. — 

1.  Prepare  nutrient  gelatine   (vide  page   145,   sections   i 
to  8). 

2.  Add    sterile    litmus    solution,    sufficient    to    tint    the 
medium  a  deep  lavender  colour. 

3.  Tube,  and  sterilise  as  for  nutrient  gelatine. 
Lactose  Litmus  Gelatine  (Wurtz).— 

1.  Prepare  nutrient  gelatine  (vide  page  145,  sections  i  to 

4). 

2.  Render  the  reaction  of  the  medium  mass  — 5. 


NUTRIENT    AGAR-AGAR.  149 

3.  Replace  in  the  steamer  at  100°  C.  for  twenty  minutes. 

4.  Clarify  with  egg  as  for  gelatine. 

5.  Weigh  out  lactose,  20  grammes  (=  2  per  cent.),  and 
dissolve  it  in  the  medium. 

6.  Filter  through  papier  Chardin. 

7.  Add  sufficient    sterile  litmus  solution  to  colour  the 
medium  pale  lavender. 

8.  Tube,  and  sterilise  as  for  nutrient  gelatine. 
Carbolised  Gelatine.— 

1.  Prepare  nutrient  gelatine  (vide  page   145,  sections   i 
to  7). 

2.  Weigh  out  carbolic  acid,  5  grammes  (=  0.5  per  cent.), 
and  dissolve  it  in  the  gelatine. 

3.  Filter  through  papier  Chardin. 

4.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

One  or  2.5  grammes  of  carbolic  acid  (=0.1  per  cent,  or 
0.25  per  cent.)  are  occasionally  used  in  place  of  the  5  grammes 
to  meet  special  requirements. 


Nutrient  Agar=agar.— 

1.  Measure  out  meat  extract,  600  c.c.,  into  a  2-litre 
flask. 

2.  Weigh  out  Witte's  peptone,   10  grammes   (—   i 
per  cent.),  salt,  5  grammes  (=  0.5  per  cent.),  and  mix 
them  into  a  smooth  paste  with  200  c.c.  meat  extract 
previously  heated  to  60°  C.;  add  the  emulsion  to  the 
meat  extract  in  the  flask. 

3.  Weigh  out  that  quantity  of  powdered  agar  neces- 
sary to  secure  the  required  percentage, — usually   15 
to  20  grammes  (—  1.5  to  2  per  cent.), — and  mix  it  into 
a  smooth  paste  with  200  c.c.  of  meat  extract  (which 
must  be  quite  cold,  as  the  agar  powder  undergoes  con- 
siderable expansion  when  first  mixed  with  hot  fluids 
and  before  solution  takes  place);  add  the  emulsion  to 
the  meat  extract,  etc.,  in  the  flask. 

4.  Place  the  flask  in  the  steamer  at  100°  C.  until  the 
agar  is  completely  dissolved.     This  will  take  about 
ninety  minutes. 

5.  Estimate  the  reaction  of  the  medium  mass;  con- 
trol the  result;  then  add  sufficient  soda  solution  to 


150  CULTURE   MEDIA. 

render  the  reaction  of  the  calculated  bulk  of  medium 
+ 10.     (See  Agar  Expansion  Table,  page  135.) 

6.  Replace  in  the  steamer  for  twenty  minutes  (to 
complete  the  precipitation  of  the  phosphates,  etc.). 

7.  Allow  the  medium  mass  to  cool  to  60°  C.     Well 
whip  the  whites  of  two  eggs,  add  to  the  contents  of  the 
flask,  and  replace  in  the  steamer  at  100°  C.  for  about 
half  an  hour  (until  the  egg-albumen  has  coagulated 
and  formed  large,  firm  masses  floating  on  and  in  clear 
agar). 

8.  Filter  through  papier  Chardin,  by  the  aid  of  a 
hot- water  funnel  (Fig.  83)  into  a  sterile  flask. 

9.  Tube,  and  sterilise  in  the  steamer  at  100°  C.  for 
thirty  minutes  on  each    of  three  consecutive  days — 
i..e.,  by  the  discontinuous  method. 

Brain  Agar;  Spleen  Agar. — Use  meat  extract  pre- 
pared from  sheep  (or  ox)  brain  and  spleen  respectively, 
and  proceed  exactly  as  if  making  nutrient  agar  (vide 
supra). 

Rapid  Method  of  Preparing  Nutrient  Agar. — 

1.  Finely  mince  500  grammes  of  lean  beef  and  add 
to  800  c.c.  of  distilled  water  in  a  flask;  place  the  flask 
in  a  water-bath,  and  raise  the  temperature  of  its  con- 
tents to  and  keep  at  45°  C.  for  twenty  minutes;  then 
rapidly  raise  the  temperature  to  100°  C.,  and  maintain 
there  for  ten  minutes. 

2.  Weigh  a  2 -litre  flask  and  note  the  weight — or  coun- 
terpoise exactly.     Filter  the  mixture  into  the  flask 
and  again  weigh,  but  do  not  make  up  the  filtrate  to 
1000  c.c.  as  in  the  ordinary  method. 

3.  Weigh  out  and  mix  10  grammes  of  peptone,  5 
grammes  of  salt,  and  20  grammes  of  powdered  agar, 
and  make  into  a  thick  paste  with   150  c.c.  distilled 
water,  and  add  to  the  meat  extract  in  the  flask;  re- 
turn the  flask  to  the  water-bath. 

4.  Arrange  a  lo-litre  tin  can  (with  copper  bottom, 
such  as  is  used  in  the  preparation  of  distilled  water) 


BRAIN  AGAR — SPLEEN  AGAR.          151 

by  its  side,  fill  the  can  with  boiling  water,  and  place 
a  lighted  Bunsen  burner  under  it.  Fit  a  long  safety 
tube  to  the  neck  of  the  can;  also  a  delivery  tube,  bent 
twice  at  right  angles  and  adjusted  to  reach  to  the  bot- 
tom of  the  interior  of  the  flask.  (See  also  Rapid  Method 
of  Preparing  Gelatine,  page  145.) 

5.  Keep  the  water  in  the  can  vigourously  boiling,  and 
so  steam  at  100°  C.,  bubbling  through  the  medium 
mass,  for  twenty-five  minutes,  by  which  time  com- 
plete solution  of  the  agar  is  effected. 

6.  Now    weigh    the   flask    and    its    contents;    then 
(1035  l  grammes  +  weight  of  flask)  minus  (weight  of 
flask  and  its  contents)  equals  the  weight  of  water  re- 
quired to  make  up  the  bulk  of  the  medium  to  i  litre. 
Add  the  requisite  amount. 

7.  Titrate,  and  estimate  the  reaction  of  the  medium 
mass;   control   the  result.     Calculate  the  amount   of 
soda  solution  required  to  make  the  reaction  of  the 
medium  mass  +10  (i.  e.,  calculated  for  1000  c.c.,  less 
the  quantity  used  for  the  titrations). 

8.  Add  the  necessary  amount  of  soda  solution  and 
heat  in  the  steamer  at  100°  C.  for  twenty  minutes. 

9.  Clarify  with  egg,  filter,  tube,  and  sterilise  as  for 
nutrient  agar  (vide  page  150), 

Glycerine  Agar. — 

1.  Prepare  nutrient  agar  (vide  page  149,  sections  i  to  8). 
Measure  out  1000  c.c. 

2.  Measure  out  pure  glycerine,  60  c.c.    (=  6  per  cent.), 
and  add  to  the  agar. 

3.  Tube,  and  sterilise  as  for  nutrient  agar. 
Sugar  Agar.— 

1.  Prepare  nutrient  agar  (vide  page  149,  sections  i  to  8). 
Measure  out  1000  c.c. 

2.  Weigh  out  glucose,  20  grammes  (=  2  per  cent.),  and 
dissolve  in  the  clear  agar. 

3.  Tube,  and  sterilise  as  for  nutrient  agar. 

NOTE. — In  certain  cases,  lactose,  maltose,  or  saccharose 
(in  similar  percentage)  is  substituted  for  glucose. 

1  Compare  note  on  page  147.     The  average  weight  of  I  litre  of  2  per  cent, 
nutrient  agar  when  completed  is  1010.5  grammes. 


152  CULTURE  MEDIA. 

Glucose  Formate  Agar  (Kitasato). — 

1.  Prepare  nutrient  agar  (vide  page  149,  sections  i  to  8). 
Measure  out  1000  c.c. 

2.  Weigh    out    glucose,    20    grammes    (—  2    per   cent.), 
sodium  formate,  4  grammes  (=  0.4  per  cent.),  and  dissolve 
in  the  agar. 

3.  Tube,  and  sterilise  as  for  nutrient  agar. 
Sulphindigotate  Agar.— 

1.  Prepare  nutrient  agar  (vide  page  149,  sections  i  to  8). 
Measure  out  1000  c.c. 

2.  Weigh    out    glucose,    20    grammes    (--  2    per    cent.), 
sodium  sulphindigotate,  i   gramme   (— o.i  percent.),  and 
dissolve  in  the  hot  agar. 

3.  Tube,  and  sterilise  as  for  nutrient  agar. 
Lactose  Litmus  Agar  (Wurtz). — 

1.  Prepare  nutrient  agar  (vide  page  149,  sections  i  to  4). 

2.  Render  the  reaction  of  the  medium  mass  — 5. 

3.  Replace  in  the  steamer  at  100°  C.  for  twenty  minutes. 

4.  Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient 
agar. 

5.  Weigh  out  lactose,  20  grammes  (—  2  per  cent.),  and 
dissolve  it  in  the  medium. 

6.  Filter  through   papier   Chardin,    using  the  hot- water 
funnel. 

7.  Add  sterile  litmus  solution,   sufficient  to  colour  the 
medium  a  pale  lavender. 

8.  Tube,  and  sterilise  as  for  nutrient  agar. 
Carbolised  Agar. — 

i;  Prepare  nutrient  agar  (vide  page  149,  sections  i  to  8). 
Measure  out  900  c.c. 

2.  Weigh  out  i  gramme  pure  phenol  and  dissolve  in  the 
medium. 

3.  Tube,  and  sterilise  as  for  nutrient  agar. 

Gelatine  Agar. — This  medium  is  prepared  by  adding  to 
nutrient  gelatine  sufficient  agar  to  ensure  the  solidity  of 
the  medium  when  incubated  at  temperatures  above  22°  C. 
If  it  is  intended  to  employ  an  incubating  temperature  of 
30°  C.,  10  per  cent,  gelatine  and  0.5  per  cent,  agar  must  be 
dissolved  in  the  meat  extract  before  the  addition  of  the  pep- 
tone and  salt;  while  for  incubating  at  37°  C.,  12  per  cent, 
gelatine  and  0.75  per  cent,  agar  must  be  used.  Avoid  the 
addition  of  more  agar  than  is  absolutely  necessary,  other- 
wise the  action  upon  the  medium  of  such  organisms  as  elabo- 
rate a  liquefying  ferment  may  be  retarded  or  completely 
inhibited. 

i.  Measure  out  600  c.c.  meat  extract  into  a  2-litre  flask, 
and  add  to  it  gelatine,  100  grammes  (or  120,  as  may  be 
necessary) . 


BLOOD-SERUM.  153 

2.  Weigh  out  powdered  agar,  5  grammes  (or  7.5  grammes, 
as  required),  emulsify  with  200  c.c.  meat  extract,  and  add 
to  the  contents  of  the  flask. 

3.  Heat  in  the  steamer  at  100°  C.  for  ninety  minutes  to 
completely  dissolve  the  agar  and  gelatine. 

4.  Weigh  out  peptone,  10  grammes,  salt,  5  grammes ;  emul- 
sify with  200  c.c.  meat  extract  previously  heated  to  60°  C., 
and  add  to  the  contents  of  the  flask. 

5.  Replace  in  the  steamer  for  fifteen  minutes. 

6.  Estimate  the  reaction;  control  the  result.     Then  add 
sufficient  caustic  soda  solution  to  render  the  reaction  -j-io. 

7.  Replace  in  the  steamer  at  100°  C.  for  twenty  minutes. 

8.  Cool  to  60°  C.     Clarify  with  egg  as  for  nutrient  gela- 
tine. 

9.  Filter  through  papier  Chardin,   using  the  hot- water 
funnel. 

10.  Tube,   and  sterilise  in  the  steamer  at    100°  C.   for 
thirty  minutes  on  each  of  three  consecutive  days. 

Agar  Gelatine  (Guarniari).— 

1.  Measure  out  meat  extract,  750  c.c.,  into  a  2-litre  flask, 
and  add  to  it  gelatine,  50  grammes. 

2.  Weigh  out  powdered  agar,  3  grammes;  emulsify  with 
cold  distilled  water,  50  c.c.,  and  add  to  the  contents  of  the 
flask. 

3.  Heat  in  the  steamer   at    100°  C.  for  sixty  to   ninety 
minutes  to  completely  dissolve  the  agar  and  gelatine. 

4.  Weigh    out    Witte's    peptone,    25    grammes,    salt,    5 
grammes,   and  emulsify  with   200  c.c.   meat  extract  pre- 
viously heated  to  60°  C.,  and  add  to  the  contents  of  the 
flask. 

5.  Replace  in  the  steamer  for  fifteen  minutes. 

6.  Neutralise  carefully  to  litmus  paper  by  the  successive 
additions  of  small  quantities  of  normal  soda  solution. 

7.  Replace  in  the  steamer  at  100°  C.  for  twenty  minutes. 

8.  Cool  to  60°  C.     Clarify  with  egg  as  for  nutrient  gela- 
tine. 

9.  Filter  through  papier  Chardin,   using  the  hot- water 
funnel. 

10.  Tube,  and  sterilise  in  the  steamer  at  100°  C.  for  thirty 
minutes  on  each  of  three  consecutive  days. 


Blood=serum. — 

i.  vSterilise  cylindrical  glass  jar  (Fig.  90)  and  its 
cover  by  dry  heat,  or  by  washing  first  with  ether  and 
then  with  alcohol. 


154 


CULTURE  MEDIA. 


2.  Collect  blood  from  sheep  or  ox  in  the  sterile  cylin- 
der. 

3.  Allow  the  vessel  to  stand  for  fifteen  minutes  for 
the  blood  to  coagulate.     (This  must  be  done  before 
leaving  the  slaughter-house,  otherwise  the  serum  will 
be  stained  with  haemoglobin.) 

4.  Separate  the  clot  from  the  sides  of  the  vessel  by 
means  of  a  sterile  glass  rod  (the  yield  of  serum  is  much 
smaller  when  this  is  not  done),  and  place  the  cylinder 
in  the  ice-chest  for  twenty-four  hours. 


Fig.  90. — Blood- serum  jar  with  wicker  basket  for  transport. 

5.  Remove  the  serum  with  sterile  pipettes,  or  syphon 
it  off,  and  fill  into  sterile  tubes  (5  c.c.  in  each)  or  flasks. 

6.  Sterilise  the  serum  by  the  fractional  method — 
that  is,  by  exposure  in  a  water-bath  to  a  temperature 
of  56°  C.  for  half  an  hour  on  each  of  six  consecutive 
days;  store  in  the  fluid  condition. 

6a.  Or  heat  tubes  containing  serum  to  56°  C.  in  a 
water-bath  for  half  an  hour  on  each  of  two  successive 
days. 


GLYCERINE  BLOOD-SERUM. 

7.  On  the  third  day,  heat  the  tubes,  in  a  sloping 
position,  in  a  serum  inspissator  to  about  72°  C.  (A 
coagulum  is  formed  at  this  temperature  which  is  fairly 
transparent;  above  72°  C.,  a  thick-  turbid  coagulum  is 
formed.) 

The  serum  inspissator  (Fig.  9 1 )  in  its  simplest  form 
is  a  double-walled  rectangular  copper  box,  closed 
in  by  a  loose  glass  lid,  and  cased  in  felt  or  asbestos— 
the  space  between  the  walls  is  filled  with  water.  The 
inspissator  is  supported  on  adjustable  legs  so  that  the 


Fig.  91. — Serum  inspissator. 

serum  may  be  solidified  at  any  desired  "  slant,"  and 
is  heated  from  below  by  a  Bunsen  burner.  The  more 
elaborate  forms  resemble  the  hot-air  oven  (Fig.  18) 
in  shape  and  are  provided  with  thermo-regulators. 

8.  Place  the  tubes  in  the  incubator  at  37°  C.  for 
forty-eight  hours  in  order  to  eliminate  those  that  have 
been  contaminated.  Store  the  remainder  in  a  cool 
place  for  future  use. 

Glycerine  Blood-serum.— 

i.  Prepare  blood-serum  as  described  above,  sections  i 
to  4. 


156  CULTURE   MEDIA. 

2.  Add  6  per  cent,  pure  glycerine. 

3.  Complete  as  described  above  for  ordinary  blood-serum, 
sections  5  to  7. 

NOTE. — Different  percentages  of  glycerine — from  4  per 
cent,  to  8  per  cent.-1— are  used  for  special  purposes.  Five 
per  cent,  is  that  usually  employed. 

Blood-serum  (Lbffler).— 

1.  Prepare  nutrient  bouillon  (vide  page  141),  using  meat 
extract  made  from  veal  instead  of  beef. 

2.  Add  i  per  cent,  glucose  to  the  bouillon,  and  allow  it 
to  dissolve  completely. 

3.  Now  add  300  c.c.  clear  blood-serum  (vide  page  153, 
sections  i  to  4)  to  every  100  c.c.  of  this  bouillon. 

4.  Fill  into  sterile  tubes  and  complete  as  for  ordinary 
blood-serum. 

Blood-serum  (Lorrain  Smith). — 

1.  Collect  blood-serum  (vide  page  153,  sections  i  to  4), 
as  free  from  haemoglobin  as  possible. 

2.  Weigh  out  0.15  per  cent,  sodium  hydrate  and  dissolve 
it  in  the  fluid  (or  add  0.375  c.c.  of  dekanormal  soda  solution 
for  every  100  c.c.  of  serum). 

3.  Tube,  and  stiffen  at  100°  C.  in  the  serum  inspissator. 

4.  Incubate  at  37°  C.  for  forty-eight  hours  to  eliminate 
any  contaminated  tubes.     Store  the  remainder  for  future 
use. 

Ascitic  Bouillon  (Serum  Bouillon).— 

1.  Collect  ascitic   fluid    (pleuritic   fluid,   hydrocele  fluid, 
etc.,  by  aspiration  directly  into  sterile  flasks,  under  strict 
aseptic  precautions. 

2.  Mix  the  serum  with  twice  its  bulk  of  sterile  nutrient 
bouillon  (vide  page  141). 

3.  If  considered  necessary  (on  account  of  the  presence  of 
blood,  crystals,  etc.),  filter  the  serum  bouillon  through  porce- 
lain filter  candle. 

4.  Tube,  and  sterilise  by  the  fractional  method  for  half 
an  hour  on  each  of  five  consecutive  days. 

5.  Incubate  at  37°  C.  for  forty-eight  hours  and  eliminate 
contaminated  tubes.     Store  for  future  use. 

Serum  Agar  (Wertheimer). — 

1.  Prepare  nutrient  agar  (vide  page  149),  to  the  following 
formula:  agar,  2  per  cent.;  peptone,  2  per  cent.;  salt,  0.5 
per  cent. ;  meat  extract,  quantum  sufficit. 

2.  Make  reaction  of  medium  -f- 10. 

3.  Filter;  tube  in  quantities  of  5  c.c. 

4.  Sterilise  by  the  discontinuous  method. 

5.  After  the  last  sterilisation  cool  to  42°  C.,   then  add 
5  c.c.  sterile  blood-serum  from  human  placenta  (sterilised, 


SERUM   AGAR.  157 

if  necessary,  by  the  fractional  method)  to  each  tube;  slope 
the  tubes. 

6.  When  set,  incubate  at  37°  C.  for  forty-eight  hours,  to 
eliminate  any  contaminated  tubes.  Store  the  remainder  for 
future  use. 

Serum  Agar  (Heiman).— 

1.  Prepare  nutrient  agar  (vide  page  149),  to  follow- 
ing formula:  agar,  2  per  cent.;  peptone,  1.5  per  cent.; 
salt,  0.5  per  cent.;  glucose,  2  per  cent;    meat  extract, 
quantum  sufficit. 

2.  Make  reaction  of  medium  +10. 

3.  Filter;  tube  in  quantities  of  6  c.c. 

4.  Sterilise  by  the  discontinuous  method. 

5.  After  the   third   sterilisation   cool   the  tubes   to 
42°  C.,  and  add  to  each  3  c.c.  of  sterile  hydrocele  fluid, 
ascitic  fluid,  or  pleuritic  effusion  (previously  sterilised, 
if   necessary,  by    the   fractional   method);    allow  the 
tubes  to  solidify  in  a  sloping  position. 

6.  When  solid,  incubate  at  37°  C.  for  forty-eight 
hours,  and  eliminate  any  contaminated  tubes.     Store 
the  remainder  for  future  use. 

Serum  Agar  (Kanthack  and  Stevens). — 

1.  Collect  ascitic,  pleuritic,  or  hydrocele  fluid  in  sterile 
flasks  and  allow  it  to  stand  in  the  ice-chest  for  twelve  hours 
to  sediment. 

2.  Decant  the  clear  fluid  into  a  measuring  cylinder. 

3.  Add  0.5  c.c.  dekanormal  NaOH  solution  for  every  100 
c.c.  serum,  and  mix  thoroughly. 

4.  Heat  in  the  steamer  for  sixty  minutes. 

5.  Weigh  out  15  grammes  agar,  emulsify  with  about  200 
c.c.  of  the  alkaline  fluid,  and  add  to  the  remainder  of  the 
fluid. 

6.  Heat  in  the  steamer  ninety  minutes  to  dissolve  the 
agar. 

7.  Filter  through  papier  Chardin,  using  a  hot- water  funnel. 

8.  Weigh  out  glucose,  i  per  cent.,  and  dissolve  it  in  the 
clear  agar. 

8a.  If  desired,  add  glycerine,  5  per  cent.,  to  the  clear 
agar. 

9.  Tube,  and  sterilise  in  the  steamer  at  100°  C.  for  thirty 
minutes  on  each  of  three  consecutive  days. 


158  CULTURE    MEDIA. 

Blood  Agar  (Washbourn).— 

1.  Melt  up  several  tubes  of  nutrient  agar  (vide  page 
149)  and  allow  them  to  solidify  in  the  oblique  position. 

2.  Place  the  tubes,   in  the  horizontal  position,   in 
the  "  hot"  incubator  for  forty-eight  hours,  to  evaporate 
off  some  of  the  condensation  water. 

3.  Kill  a  small  rabbit  with  chloroform  and  nail  it  out 
on  a  board  (as  for  a  necropsy).     Moisten  the  hair  thor- 
oughly with  2  per  cent,  solution  of  lysol. 

4.  Sterilise  several  pairs  of  forceps,  scissors,  etc.,  by 
boiling. 

5.  Reflect  the  skin  over  the  thorax  with  sterile  in- 
struments. 

6.  Open  the  thoracic  cavity  by  the  aid  of  a  fresh  set 
of  sterile  instruments. 

7.  Open  the  pericardium  with  another  set  of  sterile 
instruments. 

8.  Sear  the  surface  of  the  left  ventricle  with  a  red- 
hot  iron  and  remove  fluid  blood  from  the  heart  by 
means   of  sterile  pipettes  (e.  g.,  those  shown  in  Fig. 
10,  c). 

9.  Deliver  a  small  quantity  of   the   blood   on   the 
slanted  surface  of  the  agar  in  each  of  the  tubes,  and 
allow  it  to  run  over  the  entire  surface  of  the  medium. 

10.  Place  the  tubes  in  the  horizontal  position  and 
allow  the  blood  to  coagulate. 

11.  Return  the  " blood  agar"  to  the  hot  incubator 
for  forty-eight  hours  and  eliminate  any  contaminated 
tubes.     Store  the  remainder  for  future  use. 

Urine  Media.— 

1.  Collect  freshly  passed  urine  in  sterile  flask  or  flasks. 

2.  Place  the  flask  in  the  steamer  at   100°  C.  for  thirty 
minutes. 

3.  Filter  through  two  thicknesses  of  Swedish  filter  paper. 

4.  Tube,  and  sterilise  by  the  discontinuous  method. 
(Leave  the  reaction  unaltered.) 

Urine  Gelatine. — 

i.  Collect  freshly  passed  urine  in  sterile  flask. 


URINE    AGAR.  159 

2.  Take  the  specific  gravity,  and,  if  above  1010,  dilute 
with  sterile  water  until  that  point  is  reached. 

3.  Estimate  (with  control)  at  the  boiling-point,  and  note 
the  reaction  of  the  urine. 

4.  Weigh  out  gelatine,  10  per  cent.,  and  add  to  the  urine 
in  the  flask. 

5.  Heat  in  the  steamer  at  100°  C.  for  one  hour  to  dissolve 
the  gelatine. 

6.  Estimate  the  reaction  and  add  sufficient  caustic  soda 
solution  to  restore  the  reaction  of  the  medium  mass  to  the 
equivalent  of  the  original  urine. 

7.  Cool  to  60°  C.   and  clarify  with  egg  as  for  nutrient 
gelatine  (vide  page  145). 

8.  Filter  through  papier  Chardin. 

9.  Tube,  and  sterilise  as  for  gelatine. 
Urine  Gelatine  (Heller).— 

1.  Collect  freshly  passed  urine  in  sterile  flask. 

2.  Filter  through  animal  charcoal  to  remove  part  of  the 
colouring  matter. 

3.  Take  the  specific  gravity,  and  if  above   1010,  dilute 
with  sterile  water  till  this  gravity  is  reached. 

4.  Add  Witte's  peptone,  i  per  cent.;  salt,  0.5  per  cent.; 
gelatine,  10  per  cent. 

5.  Heat  in  the  steamer  at  100°  C.  for  one  hour,  to  dissolve 
the  gelatine,  etc. 

6.  Add  normal  caustic  soda  solution  in  successive  small 
quantities,  and  test  the  reaction  from  time  to  time  with 
litmus  paper,  until  the  fluid  reacts  faintly  alkaline. 

7    Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient 
gelatine  (vide  page  145). 

8.  Filter  through  papier  Chardin. 

9.  Tube,  and  sterilise  as  for  nutrient  gelatine. 

Urine  Agar. — 

1.  Collect  freshly  passed  urine  in  sterile  flask. 

2.  Weigh  out  1.5  per  cent,  or  2  per  cent,  powdered 
agar,  and  add  it  to  the  urine. 

3.  Heat  in  the  steamer  ninety  minutes  to  dissolve 
the  agar. 

4.  Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient 
agar  (vide  page  150). 

5.  Filter  through  papier   Chardin,   using   the   hot- 
water  funnel. 

6.  Tube,  and  sterilise  as  for  nutrient  agar. 
(Leave  the  reaction  unaltered.) 


160  CULTURE    MEDIA. 

Egg=albumen. — 

1.  Break  several  fresh  eggs  (hens',  ducks',  or  turkeys' 
eggs),  and  collect  the  "  whites"  in  a  graduated  cylinder, 
taking  care  to  avoid  admixture  with  the  yolks. 

2.  Add  40  per  cent,  distilled  water,  and  incorporate 
the  mixture  thoroughly  by  the  aid  of  an  egg-whisk. 

3.  Weigh  out  0.15  per  cent,  sodium  hydrate  and 
dissolve  it  in  the  fluid  (or  add  the  amount  of  dekanormal 
caustic  soda  solution  calculated  to  yield  the  required 
percentage  of  soda  in  the  total  bulk  of  the  fluid— i.  e., 
0.375  c-c-  °f  dekanormal  NaOH  solution  per  100  c.c. 
of  the  mixture). 

3<z.  Glucose  to  the  extent  of  i  to  2  per  cent,  may  now 
be  added,  if  desired. 

4.  Strain  the  mixture  through  butter  muslin   and 
filter  through  a  porcelain  filter  candle  into  a  sterile 
filter  flask. 

5.  Tube,  and  stiffen  at  100°  C.  in  the  serum  inspis- 
sator. 

6.  Incubate   at   37°   C.    for   forty-eight   hours   and 
eliminate  any  contaminated  tubes ;  store  the  remainder 
for  future  use. 

Egg-albumen  (Tarchanoff  and  Kolesnikoff). — 

1.  Place  unbroken  hens'  eggs  in  dekanormal  caustic  soda 
solution  for  ten  days.     (After  this  time  the  white  becomes 
firm  like  gelatine.) 

2.  Carefully  remove  the  shell  and  cut  the  egg  into  fine 
slices. 

3.  Wash  for  two  hours  in  running  water. 

4.  Place  the  egg  slices  in  a  large  beaker  and  sterilise  in 
the  steamer  at  100°  C.  for  one  hour. 

5.  Transfer  each  slice  of  egg  by  means  of  a  pair  of  steril- 
ised forceps  to  a  Petri  dish  or  large  capsule. 

6.  Sterilise  in  the  steamer  at  100°  C.  for  twenty  minutes 
on  each  of  three  consecutive  days. 

Milk.- 

1.  Pour  i  litre  of  fresh  milk  into  a  large  separator 
funnel,  and  heat  in  the  steamer  at  100°  C.  for  one  hour. 

2.  Remove  from  the  steamer  and  estimate  the  re- 


WHEY    GELATINE.  l6l 

action  of  the  milk  (normal  cows'  milk  averages  +17). 
If  of  higher  acidity  than  +20,  or  lower  than  +10,  re- 
ject this  sample  of  milk  and  proceed  with  another  litre 
of  milk  from  a  different  source. 

Carefully  eliminate  milk  to  which  antiseptics  have 
been  added  as  preservatives. 

3.  Allow  the  milk  to  cool,  when  the  fat  or  cream 
will  rise  to  the  surface  and  form  a  thick  layer. 

4.  Draw  off  the  subnatant  fat-free  milk  into  sterile 
tubes  (10  c.c.  in  each). 

5.  Sterilise  in  the  steamer  at   100°  C.  for  twenty 
minutes  on  each  of  five  successive  days. 

6.  Incubate   at   37°   C.    for   forty-eight   hours   and 
eliminate    any    contaminated    tubes.     Store    the    re- 
mainder for  future  use. 

Litmus  Milk.— 

1.  Prepare  milk  as  described  above,  sections  i  to  3. 

2.  Draw  off  the  fat-free  milk  into  a  flask. 

3.  Add  sterile  litmus  solution,  sufficient  to  colour 
the  milk  a  deep  lavender. 

4.  Tube,  sterilise,  etc.,  as  for  milk. 

Litmus  Whey.— 

1.  Curdle  fresh  milk  by  adding  rennet  (or  by  acidifying 
with  hydrochloric  acid). 

2.  Filter  off  the  whey  into  a  sterile  flask. 

3.  Heat  in  the  steamer  at  100°  C.  for  one  hour. 

4.  Filter  into  a  sterile  flask. 

5.  Tint  the  whey  with  litmus  solution  to  a  deep  purple 
red. 

6.  Tube,  and  sterilise  as  for  milk. 
Whey  Gelatine.— 

1.  Curdle   fresh   milk   by   adding  rennet;   filter   off  the: 
whey  into  a  sterile  flask. 

2.  Estimate  and  note  the  reaction  of  the  whey. 

3.  Weigh  out  gelatine,   10  per  cent.,  and  add  it  to  the 
whey  in  the  flask. 

4.  Heat  in  the  steamer  at  100°  C.  for  one  hour  to  dissolve 
the  gelatine. 

5.  Estimate  the  reaction  of  the  medium  mass;  then  add 
sufficient  caustic  soda  solution  to  restore  the  reaction  of  the 
medium  mass  to  the  equivalent  of  the  original  whey. 

ii 


1 62  CULTURE    MEDIA. 

6.  Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient 
gelatine  (vide  page  145). 

7.  Filter  through  papier  Chardin. 

8.  Tube,  and  sterilise  as  for  nutrient  gelatine. 
Whey  Agar.— 

1 .  Curdle  fresh  milk  by  adding  rennet ;  filter  off  the  whey 
into  a  sterile  flask. 

2.  Weigh  out  agar,  1.5  or  2  per  cent.,  and  add  it  to  the 
whey  in  the  flask. 

3.  Heat  in  the  steamer  at  100°  C.  for  one  hour,  to  dis- 
solve the  agar. 

4.  Cool  to  60°  C. ;  clarify  with  egg  as  for  nutrient  agar 
(vide  page   150). 

5.  Filter  through   papier   Chardin,   using  the  hot-water 
funnel. 

6.  Tube,  and  sterilise  as  for  nutrient  agar. 
Fish  Bouillon.— 

1 .  Weigh  out  herring,  mackerel,  or  cod,  500  grammes,  and 
place  in  a  large  porcelain  beaker  (or  enamelled  iron  pot). 

2.  Weigh  out  sodium  chloride,  26.5  grammes;  potassium 
chloride,  0.75  gramme;  magnesium  chloride,  3.25  grammes; 
and  dissolve  in  1000  c.c.  distilled  water.     Add  the  solution 
to  the  fish  in  the  beaker. 

3.  Place  the  beaker  in  a  water-bath  and  proceed  as  if 
preparing  meat  extract — i.   e.,   heat  gently  at  40°  C.   for 
twenty  minutes,  then  rapidly  raise  the  temperature  to,  and 
maintain  at,  the  boiling-point  for  ten  minutes. 

4.  Strain  the  mixture  through  butter  muslin  into  a  clean 
flask. 

5.  Weigh  out  peptone,   5   grammes,   and  emulsify  with 
about  200  c.c.  of  the  hot  fish  water;  incorporate  thoroughly 
with  the  remainder  of  the  fish  water  in  the  flask. 

6.  Heat  in  the  steamer  at   100°  C.  for  twenty  minutes 
to  complete  the  solution  of  the  peptone. 

7.  Filter  through  Swedish  filter  paper. 

8.  When  the  fish  bouillon  is  cold,  make  up  to  1000  c.c. 
by  the  addition  of  distilled  water  (to  replace  the  loss  from 
evaporation). 

As  an  alternative  method  "  Marvis  "  fish  food  (16  grammes)  may  be  sub- 
stituted for  the  5°o  grammes  of  fresh  fish. 

Fish  Gelatine.— 

1.  Measure  out  fish  bouillon,  1000  c.c.,  into  a  2 -litre  flask. 

2.  Add  sheet  gelatine,  100  grammes,  cut  into  small  pieces. 

3.  Heat  in  the  steamer  at  100°  C.  for  one  hour. 

4.  Cool  to  below  60°  C.,  and  clarify  with  egg. 

5.  Filter  through  papier  Chardin. 


POTATO. 


I63 


6.  Tube,  and  sterilise  as  for  nutrient  gelatine. 
Shake   well   after   the  final  sterilisation,    to    aerate   the 
medium. 

Fish  Gelatine— Agar.— 

1.  Weigh  out  powdered  agar,  5  grammes,  and  emulsify  it 
with  200  c.c.  fish  bouillon. 

2.  Wash  the  emulsion  into  a  2 -litre  flask  with  800  c.c. 
fish  bouillon. 

3.  Weigh  out  sheet  gelatine,  70  grammes,  cut  it  into  small 
pieces  and  add  it  to  the  contents  of  the  flask. 

4.  Heat  in  the  steamer  at  100°  C.  for  one  hour. 

5.  Cool  to  below  60°  C.  and  clarify  with  egg. 

6.  Filter  through  papier  Chardin. 

7.  Tube,  and  sterilise  as  for  fish  gelatine. 

Shake   well   after   the   final   sterilisation,  to    aerate   the 
medium. 


Potato.- 

1.  Choose  fairly  large  potatoes,  wash  them  well,  and 
scrub  the  peel  with  a  stiff  nail-brush. 

2.  Peel  and  take  out  the  eyes. 

3.  Remove  cylinders  from  the   longest 
diameter  of  each  potato  by  means  of  an 
apple-corer  or  a  large  cork-borer  (i.  e.,  one 
of  about  1.4  cm.  diameter). 

3a.  The  reaction  of  the  fresh  potato  is 
strongly  acid  to  phenolphthalein.  If,  there- 
fore, the  potatoes  are  required  to  approxi- 
mate +  10,  as  for  the  cultivation  of  some 
of  the  vibrios,  the  cylinders  should  be 
soaked  in  a  i  per  cent,  solution  of  sodium 
carbonate  for  thirty  minutes. 

4.  Cut  each  cylinder  obliquely  from  end 
to  end,  forming   two  wedge-shaped   por- 
tions. 

5.  Place  a  small  piece  of  sterilised  cot- 
ton-wool, moistened  with  sterile  water,  at 
the  bottom  of  a  sterile  test-tube;  insert 
the  potato  wedge  into  the  tube  so  that  its 

base  rests  upon  the  cotton- wool.     Now  plug  the  tube 
with  cotton- wool  (Fig.  92). 


Fig.  92.— Po- 
tato tube. 


164  CULTURE    MEDIA. 

6.  Sterilise  in  the  steamer   at   100°  C.  for  twenty 
minutes  on  each  of  five  consecutive  days. 

Beet-root,  carrot,  turnip,   and    parsnip   are  prepared, 
tubed,  and  sterilised  as  potato. 
Glycerinated  Potato. — 

1.  Prepare  ordinary  potato  wedges  (vide  page  163,  sec- 
tions i  to  4). 

2.  Soak  the  wedges  in  25  per  cent,  solution  of  glycerine 
for  fifteen  minutes. 

3.  Moisten  the  cotton- wool  pads  at  the  bottom  of  the 
potato  tubes  with  a  25  per  cent,  solution  of  glycerine  instead 
of  plain  water. 

4.  Insert  a  wedge  of  potato  in  each  tube  and  replug  the 
tubes. 

5.  Sterilise  in  the  steamer  at  100°  C.  for  twenty  minutes 
on  each  of  five  consecutive  days. 

Glycerine  Potato  Broth.— 

1.  Take   i  kilo  of  potatoes,  wash  thoroughly  in  water, 
peel,  and  grate  finely  on  a  bread-grater. 

2.  Weigh  the  potato  gratings,   place  them  in  a   2 -litre 
flask,  and  add  distilled  water  in  the  proportion  of  i  c.c. 
for  every  gramme  weight  of  potato.     Allow  the  flask  to 
stand  in  the  ice-chest  for  twelve  hours. 

3.  Strain  the  mixture  through  butter  muslin  and  filter 
through  Swedish  filter  paper  into   a  graduated  cylinder. 
Note  the  amount  of  the  filtrate. 

4.  Place  the  filtrate  in  a  flask,  add  an  equal  quantity 
of  distilled  water,  and  heat  in  the  steam  steriliser  for  sixty 
minutes. 

5.  Add  glycerine,  4  per  cent.,  mix  thoroughly,  and  again 
filter. 

6.  Tube  and  sterilise  in  the  steamer  at  100°  C.  for  twenty 
minutes  on  each  of  three  consecutive  days. 

Potato  Gelatine  (Eisner).— 

1.  Take   i  kilo  of  potatoes,  wash  thoroughly  in  water, 
peel,  and  finally  grate  finely  on  a  bread-grater. 

2.  Weigh  the  potato  gratings,   place  them  in  a   2 -litre 
flask,  and  add  distilled  water  in  the  proportion  of  i  c.c.  for 
every  gramme  weight  of  potato.     Allow  the  flask  to  stand 
in  the  ice-chest  for  twelve  hours. 

3.  Strain  the  mixture  through  butter  muslin,  and  filter 
through  Swedish  filter  paper  into  a  graduated  cylinder. 

4.  Add  15  per  cent,  gelatine  to  the  potato  decoction  and 
heat  in  the  steamer  for  sixty  minutes. 

5.  Estimate  the  reaction  and  render  the  reaction  of  the 
medium  mass  +25. 


BEER    WORT.  165 

6.  Cool  the  medium  to  below  60°  C. ;  clarify  with  egg  as 
for  nutrient  gelatine  (vide  page  145). 

7.  Add  i  per  cent,  potassium  iodide  (powdered)  to  the 
medium. 

8.  Filter  through  papier  Chardin. 

9.  Tube  in  quantities  of  10  c.c. 

10.  Sterilise  in  the  steamer  at  100°  C.  for  twenty  minutes 
on  each  of  three  consecutive  days. 

Potato  Gelatine  (Goadby). — 

1.  Prepare  glycerine  potato  broth  as  above,  sections   i 
to  5. 

2.  Add  10  per  cent,  gelatine  to  the  potato  decoction  and 
heat  in  the  steamer  at  100°  C.  for  sixty  minutes. 

3.  Estimate  the  reaction  and  render  -j~5- 

4.  Cool  the  medium  to  below  60°  C.,  clarify  with  egg  as 
for  nutrient  gelatine. 

5.  Filter  through  papier  Chardin. 

6.  Tube,  and  sterilise  as  for  nutrient  gelatine. 
Hay  Infusion. — 

1.  Weigh  out  dried  hay,   10  grammes,  chop  it  up  into 
fine  particles  and  place  in  a  flask. 

2.  Add  1000  c.c.  distilled  water,  heated  to  70°  C. ;  close 
the  flask  with  a  solid  rubber  stopper. 

3.  Macerate  in  a  water-bath  at  60°  C.  for  three  hours. 

4.  Replace  the  stopper  by  a  cotton-wool  plug,  and  heat 
in  the  steamer  at  100°  C.  for  one  hour. 

5.  Filter  through  Swedish  filter  paper. 

6.  Tube,  and  sterilise  in  the  steamer  at  100°  C.  for  one 
hour  on  each  of  three  consecutive  days. 

Beer  Wort. — Wort  is  chiefly  used  as  a  medium  for 
the  cultivation  of  yeasts,  etc.,  both  in  its  fluid  form 
and  also  when  made  solid  by  the  addition  of  gelatine 
or  agar.  The  wort  is  prepared  as  follows: 

1.  Weigh  out  250  grammes  crushed  malt  and  place 
in  a  2 -litre  flask. 

2.  Add  1000  c.c.  distilled  water,  heated  to  70°  C., 
and  close  the  flask  with  a  rubber  stopper. 

3.  Place  the  flask  in  a  water-bath  regulated  to  60° 
C.  and  allow  the  maceration  to  continue  for  one  hour. 

4.  Strain  through  butter  muslin  into  a  clean  flask 
and  heat  in  the  steamer  for  thirty  minutes. 

5.  Filter  through  Swedish  filter  paper. 

6.  Tube  in  quantities  of  10  c.c.  or  store  in  flasks. 


1 66  CULTURE    MEDIA. 

7.  Sterilise  in  the  steamer  at  100°  C.  for  twenty 
minutes  on  each  of  three  consecutive  days. 

The  natural  reaction  of  the  wort  should  not  be  inter- 
fered with. 

NOTE. — It  is  sometimes  more  convenient  to  obtain 
"  unhopped  "  l  beer  wort  direct  from  the  brewery.  In 
this  case  it  is  diluted  with  an  equal  quantity  of  dis- 
tilled water,  steamed  for  an  hour,  filtered,  filled  into 
sterile  flasks  or  tubes,  and  sterilised  by  the  discon- 
tinuous method. 

Wort  Gelatine.— 

1.  Measure  out  wort  (prepared  as  above),  1000  c.c., 
into  a  sterile  flask. 

2.  Weigh  out  gelatine,  100  grammes  (=  10  percent.), 
and  add  it  to  the  wort  in  the  flask. 

3.  Heat  in  the  steamer  at  100°  C.  for  one  hour,  to 
dissolve  the  gelatine. 

4.  Cool  to  60°  C.;  clarify  with  egg  as  for  nutrient 
gelatine  (vide  page  145). 

5.  Filter  through  papier  Char  din. 

6.  Tube,  and  sterilise  as  for  nutrient  gelatine. 
Wort  Agar. — 

1.  Measure  out  wort   (as  above),   800  c.c.,  into  a 
sterile  flask. 

2.  Weigh  out  powdered  agar,  20  grammes;  mix  into 
a  smooth  paste  with  200  c.c.  of  cold  wort  and  add  to 
the  wort  in  the  flask. 

3.  Heat  in  the  steamer  at  100°  C.  for  ninety  minutes, 
to  dissolve  the  agar. 

4.  Cool  to  60°  C.;  clarify  with  egg  as  for  nutrient 
agar  (vide  page  150). 

5.  Filter  through  papier   Char  din,   using  the   hot- 
water  funnel. 

6.  Tube,  and  sterilise  as  for  nutrient  agar. 

1  "  Hopped"  wort  exerts  a  toxic  effect  upon  many  bacteria,  including  the 
lactic  acid  bacteria. 


MILK   RICE.  167 

Wine  Must. — (Wine  must  is  obtained  from  Sicily,  in 
hermetically  sealed  tins,  in  a  highly  concentrated  form, — 
as  a  thick  syrup, — but  not  sterilised.) 

1.  Weigh   out  "wine   must,"   200   grammes,   place  in  a 
2 -litre  flask  and  add  distilled  water,  800  c.c. 

2.  Weigh  out  ammonium  tartrate,  5  grammes,  and  add 
to  the  dilute  must. 

3.  Place  the  flask  in  a  water-bath  regulated  to  60°  C. 
for  one  hour  and  incorporate  the  mixture  thoroughly  by 
frequent  shaking. 

4.  Filter  through  papier  Chardin. 

5.  Tube,  and  sterilise  by  the  discontinuous  method  (three 
days). 

Wheat  Broth  (Gasperini).— 

1 .  Weigh  out  and  mix  wheat  flour,  1 50  grammes ;  magne- 
sium sulphate,  0.5  gramme;  potassium  nitrate,  i  gramme; 
glucose,  15  grammes. 

2.  Dissolve  the  mixture  in  1000  c.c.  of  water  heated  to 
100°  C. 

3.  Filter  through  papier  Chardin. 

4.  Tube,  and  sterilise  by  the  discontinuous  method. 
Bread  Paste.— 

1.  Grate  stale  bread  finely  on  a  bread-grater. 

2.  Distribute   the   crumbs   in  sterile   Erlenmeyer  flasks, 
sufficient  to  form  a  layer  about  half  an  inch  thick  over  the 
bottom  of  each. 

3.  Add  as  much  distilled  water  as  the  crumbs  will  soak 
up,  but  not  enough  to  cover  the  bread. 

4.  Plug  the  flasks  and  sterilise  in  the  steamer  at  100°  C. 
for  thirty  minutes  on  each  of  jour  consecutive  days. 

Milk  Rice  (Eisenberg). — 

1.  Measure  out  nutrient  bouillon, 70  c.c.,  and  milk,  2 10  c.c., 
and  mix  thoroughly. 

2.  Weigh  out  rice  powder,  100  grammes,  and  rub  it  up 
in  a  mortar  with  the  milk  and  broth  mixture. 

3.  Fill  the  paste  into  sterile  capsules,  spreading  it  out 
so  as  to  form  a  layer  over  the  bottom  of  each. 

4.  Heat  over  a  water-bath  at  100°  C.  until  the  mixture 
solidifies. 

5.  Replace   the   lids   of   the   capsules.     Sterilise   in   the 
steamer  at  100°  C.  for  twenty  minutes  on  each  of  three  con- 
secutive days. 

(A  solid  medium  of  the  colour  of  cafe  au  lait  is  thus  pro- 
duced.) 

Milk  Rice  (Soyka).— 

i.  Measure  out  nutrient  bouillon,  50  c.c.,  and  milk,  150 
c.c.,  and  mix  thoroughly. 


1 68  CUI/TURE    MEDIA. 

2.  Weigh  out  rice  powder,  100  grammes,  and  rub  it  up  in 
a  mortar  with  the  milk  and  broth  mixture. 

3.  Fill  the  paste  into  sterile  capsules,  to  form  a  layer  over 
the  bottom  of  each. 

4.  Replace  the  lids  of  the  capsules. 

5.  Sterilise  in  the  steamer  at  100°  C.  for  twenty  minutes 
on  each  of  three  consecutive  days. 

(A  pure  white,  opaque  medium  is  thus  formed.) 


Peptone  Water  (Dunham).— 

1.  Weigh  out  Witte's  peptone,    10  grammes,   and 
salt,  5  grammes,  and  emulsify  with  about  250  c.c.  of 
distilled  water  previously  heated  to  60°  C. 

2.  Pour  the  emulsion  into  a  litre  flask  and  make  up 
to  1000  c.c.  by  the-  addition  of  distilled  water. 

3.  Heat  in  the  steamer  at  100°  C.  for  thirty  minutes. 

4.  Filter  through  Swedish  filter  paper. 

5.  Tube  in  quantities  of  10  c.c.  each. 

6.  Sterilise  in  the  steamer  at   100°  C.  for  twenty 
minutes  on  each  of  three  consecutive  days. 

Peptone  Rosolic  Acid  Water. — 

1.  Weigh  out  rosolic  acid   (coralline),  0.5  gramme,  and 
dissolve  it  in  80  per  cent,  alcohol,  100  c.c.     Keep  this  as  a 
stock  solution. 

2.  Measure  out  peptone  water  (Dunham),   100  c.c.,  and 
rosolic  acid  solution,  2  c.c.,  and  mix. 

3.  Heat  in  the  steamer  at  100°  C.  for  thirty  minutes. 

4.  Filter  through  Swedish  filter  paper. 

5.  Tube,  and  sterilise  as  for  peptone  water. 
Iron  Peptone  Solution  (Pakes). — 

1.  Weigh  out  peptone,  30  grammes,  and  emulsify  it  with 
200  c.c.  tap  water,  previously  heated  to  about  60°  C. 

2.  Wash  the  emulsion  into  a  litre  flask  with  80  c.c.  tap 
water. 

3.  Weigh  out  salt,  5  grammes,  and  sodium  phosphate,  3 
grammes,  and  dissolve  in  the  mixture  in  the  flask. 

4.  Heat  the  mixture  in  the  steamer  at  100°  C.  for  thirty 
minutes,  to  complete  the  solution  of  the  peptone,  and  filter 
into  a  clean  flask. 

5.  Fill  into  tubes  in  quantities  of  10  c.c.  each. 

6.  Add  to  each  tube  o.i  c.c.  of  a  2  per  cent,  neutral  solu- 


SAI/T    AGAR.  169 

tion  of  ferric  tartrate.  (A  yellowish- white  precipitate 
forms.) 

7.  Sterilise  as  for  peptone  solution. 

NOTE. — A  similar  quantity  of  a  i  per  cent,  neutral  solution 
of  lead  acetate  may  be  substituted  for  the  iron  salt. 


Nitrate  Water  (Pakes). — 

1.  Weigh  out  Witte's  peptone,    10  grammes,    and 
emulsify  it  with  200  c.c.  ammonia-free  distilled  water 
previously  heated  to  60°  C. 

2.  Wash  the  emulsion  into  a  flask  and  make  up  to 
looo  c.c.,  with  similar  water. 

3.  Heat  in  the  steamer  at  100°  C.  for  twenty  minutes. 

4.  Weigh  out  sodium  nitrate,   i  gramme,  and  dis- 
solve in  the  contents  of  the  flask. 

5.  Filter  through  Swedish  filter  paper. 

6.  Tube,  and  sterilise  as  for  peptone  water. 


Bile  Salt  Broth  (MacConkey). — 

1.  Weigh  out  Witte's  peptone,  20  grammes  (=  2  per  cent.), 
and    emulsify    with    200    c.c.    distilled    water    previously 
warmed  to  60°  C. 

2.  Weigh    out     sodium    taurocholate     (commercial),    5 
grammes  (=0.5  per  cent.),  and  glucose,  5  grammes  (=0.5 
percent.),  and  dissolve  in  the  peptone  emulsion. 

3.  Wash  the  peptone  emulsion  into  a  flask  with  800  c.c. 
distilled  water,  and  heat  in  the  steamer  at  100°  C.  for  twenty 
minutes. 

4.  Filter  through  Swedish  filter  paper  into  a  sterile  flask. 

5.  Add   sterile  litmus   solution  sufficient   to   colour  the 
medium  to  a  deep  purple. 

6.  Fill,   in  quantities  of   10  c.c.,   into  tubes  containing 
small  gas  tubes  (vide  Fig.  85,  page  139),  and  sterilise  in  the 
steamer  at  100°  C.  for  twenty  minutes  on  each  of  three  con- 
secutive days. 

Bile  Salt  Agar  (MacConkey).— 

1.  Weigh  out  powdered  agar,    15   grammes   (=  1.5   per 
cent.),  and  emulsify  with  200  c.c.  cold  distilled  water. 

2.  Weigh  out  peptone,  20  grammes  (==  2  per  cent.),  and 
emulsify  with  200  c.c.  distilled  water  previously  warmed  to 
60°  C. 

3.  Mix  the  peptone  and  agar  emulsions  thoroughly. 

4.  Weigh  out   sodium    taurocholate,   5  grammes  (—  0.5 


170  CULTURE  MEDIA. 

per  cent.),  dissolve  it  in  600  c.c.  distilled  water,  and  use 
the  solution  to  wash  the  agar- pep  tone  emulsion  into  a  2 -litre 
flask. 

5.  Heat  in  the  steamer  at  100°  C.  for  sixty  minutes. 

6.  Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient 
agar  (vide  page  150). 

7.  Filter  through   papier  Chardin,   using  the  hot-water 
funnel. 

8.  Weigh  out  lactose,  10  grammes  (—  i  per  cent.),  and 
dissolve  it  in  the  agar. 

9.  If  desired,  add  saturated  aqueous  solution  of  neutral 
red,  2  c.c. 

10.  Tube,   and  sterilise  in  the  steamer  at   100°  C.  for 
twenty  minutes  on  each  of  three  consecutive  days. 

French  Proof  Agar  (Sabouraud). — 

1.  Weigh    out    peptone    (Chassaing),    7    grammes,    and 
emulsify  it  with  200  c.c.  distilled  water  previously  heated 
to  60°  C. 

2.  Weigh  out  powdered  agar,  13  grammes,  and  emulsify 
with  200  c.c.  cold  distilled  water. 

3.  Mix  the  two  emulsions  and  wash  into  a  2 -litre  flask 
with  600  c.c.  distilled  water. 

4.  Heat  in  the  steamer  for  ninety  minutes,  to  dissolve 
the  agar. 

5.  Cool  to  60°  C.  and  clarify  with  egg  as  for  nutrient 
agar  (vide  page  150). 

6.  Filter  through   papier  Chardin,   using  the  hot-water 
funnel. 

7.  Weigh  out  maltose,  38  grammes,  and  dissolve  in  the 
agar. 

8.  Tube,  and  sterilise  as  for  nutrient  agar. 

English  Proof  Agar  (Blaxall).— Substitute  Witte's  pep- 
tone for  that  of  Chassaing,  and  proceed  as  for  French  proof 
agar. 

Pasteur's  Solution. — 

1.  Weigh   out   and   mix   the   ash   from  10  grammes  of 
yeast;   ammonium  tartrate,   10  grammes;  cane  sugar,   100 
grammes. 

2.  Dissolve  the  mixture  in  distilled  water,  1000  c.c. 

3.  Tube  or  flask,  and  sterilise  by  the  discontinuous  method 
(three  days). 

Yeast  Water  (Pasteur).— 

1.  Weigh  out  pressed  yeast,  75  grammes;  place  in  a  2- 
litre  flask  and  add  1000  c.c.  distilled  water. 

2.  Heat  in  the  steamer  at  100°  C.  for  thirty  minutes. 

3.  Filter  through  papier  Chardin. 

4.  Tube  or  flask,  and  sterilise  as  for  Pasteur's  solution. 


ASPARAGIN    MEDIUM.  iyi 

Cohn's  Solution. — 

1.  Weigh  out  and  mix 

Acid  potassium  phosphate 5.00  grammes 

Tribasic  calcium  phosphate 0.50  gramme 

Magnesium  sulphate 5.00  grammes 

Ammonium  tartrate 10.00       " 

and  dissolve  in 

Distilled  water      . 1000  c.c. 

2.  Tube,  or  flask  and  sterilise  as  for  Pasteur's  solution. 
Naegelfs  Solution. — 

1.  Weigh  out  and  mix 

Dibasic  potassium  phosphate      I.o  gramme 

Magnesium  sulphate 0.2       " 

Calcium  chloride o.  i        " 

Ammonium  tartrate 10.0  grammes 

and  dissolve  in 

Distilled  water looo  c.c. 

2.  Tube  or  flask;  sterilise  as  for  Pasteur's  solution. 
Asparagin  Medium  (Uschinsky).— 

1.  Weigh   out   and   mix 

Asparagin  3.4  grammes 

Ammonium  lactate 10.0       " 

Sodium  chloride 5.0       " 

Magnesium  sulphate 0.2  gramme 

Calcium  chloride o.  I        " 

Acid  potassium  phosphate       I.o       " 

2.  Dissolve  the  mixture  in  distilled  water,  1000  c.c. 

3.  Add  glycerine,  40  c.c. 

4.  Tube,    and    sterilise    by    the    discontinuous    method 
(three  days). 

Asparagin  Medium  (Frankel  and  Voges). — 

1.  Weigh  out  and  mix 

Asparagin 4  grammes 

Sodium  phosphate  (neutral) 2       " 

Ammonium  lactate 6       " 

Sodium  chloride 5       " 

and  dissolve  in 

Distilled  water 1000  c.c. 

2.  Tube,  and  sterilise  by  the  discontinuous  method  (three 
days). 

NOTE. — Either  of  the  above  asparagin  media,  after  the 
addition  of  10  per  cent,  gelatine  or  1.5  per  cent,  agar,  may 
be  advantageously  employed  in  the  solid  condition. 


172  CULTURE  MEDIA. 

Winogradsky's    Solution    (for  Nitric  Organisms). — 

1.  Weigh  out  and  mix 

Potassium  phosphate l.o    gramme 

Magnesium  sulphate 0.5          " 

Calcium  chloride o.oi        " 

Sodium  chloride 2.00  grammes. 

and  dissolve  in 

Distilled  water      .    .    .    , looo  c.c. 

2.  Fill  into  flasks,  in  quantities  of  20  c.c.,  and  add  to 
each  a  small  quantity  of  freshly  washed  magnesium  car- 
bonate. 

3.  Sterilise  in  the  steamer  at  100°  C.  for  twenty  minutes 
on  each  of  three  consecutive  days. 

4.  Add  to  each  flask  containing  20  c.c.  solution,  2  c.c. 
of  a  sterile  2  per  cent,  solution  of  ammonium  sulphate. 

5.  Incubate  at  37°  C.  for  forty-eight  hours  and  eliminate 
any  contaminated  culture  flasks. 

Winogradsky's   Solution  (for  Nitrous  Organisms) — 

1.  Weigh  out  and  mix 

Ammonium  sulphate I  gramme 

Potassium  sulphate I       " 

and  dissolve  in 

Distilled  water looo  c.c. 

2.  Add   magnesium   carbonate,    previously   sterilised   by 
boiling. 

3.  Fill  into  flasks  and  sterilise  as  for  previous  solution. 
Silicate  Jelly  (Winogradsky). — 

1.  Weigh  out  and  mix 

Ammonium  sulphate 0,40  gramme 

Magnesium  sulphate 0.05       " 

Calcium  chloride o.oi        " 

i 

and  dissolve  in 

Distilled  water 50  c.c. 

Label — Solution  A. 

2.  Weigh  out  and  mix 

Potassium  phosphate o.  10  gramme 

Sodium  carbonate 0.60       " 

and  dissolve  in 

Distilled  water 50  c.c. 

Label — Solution  B. 


PLASTER-OF-PARIS   DISCS.  173 

3.  Weigh  out 

Silicic  acid 3.4  grammes 

and  dissolve  in 

Distilled  water loo  c.c. 

4.  Pour  the  silicic  acid  solution  into  a  large  porcelain 
basin. 

5.  Mix  equal  quantities  of  the  solutions  A  and  B;  then 
add  successive  small  quantities  of  the  mixed  salts  to  the 
silicic  acid  solution,  stirring  continuously  with  a  glass  rod, 
until  a  jelly  of  sufficiently  firm  consistence  has  been  formed. 

6.  Spread  a  layer  of  this  jelly  over  the  bottom  of  each 
of  several  large  capsules  or  "plates." 

7.  Sterilise  in  the  steamer  for  twenty  minutes  on  each 
of  three  consecutive  days. 

Plaster-of-Paris  Discs.— 

1.  Take  large  corks,  2.5  cm.  diameter,  and  roll  a  piece 
of  stiff  note-paper  round  each,  so  that  about  a  centimeter 
projects  as  a  ridge  above  the  upper  sur- 
face of  the  cork,  and  secure  in  position 

with  a  pin  (Fig.  93). 

2.  Mix    plaster-of-Paris    into    a    stiff 
paste  with  distilled  water,  and  fill  each 
of  the  cork  moulds  with  the  paste. 

3.  When  the  plaster  has  set,  remove 
the  paper  from  the  corks,  and  lift  up  the 
plaster  discs. 

4.  Place  the  plaster  discs  on  a  piece  of        Fig.  93. — Cork  and 
asbestos  board  and  sterilise  by  exposing     paper  mould  for  plas- 
in  the  hot-air  oven  to  150°  C.  for  half     ter-of- Paris  disc. 

an  hour. 

5.  Remove  the   sterile   discs  from  the  oven   by  means 
of  sterile  forceps,  place  each  inside  a  sterile  capsule,  and 
moisten  with  a  little  sterile  water. 

6.  Sterilise  in  the  steamer  at  100°  C.  for  twenty  minutes 
on  each  of  three  consecutive  days. 


XI.  INCUBATORS. 

AN  incubator  consists  essentially  of  a  chamber  for 
the  reception  of  cultivations,  etc.,  surrounded  by  a 
water  jacket,  the  walls  of  which  are  of  metal,  usually 
copper,  and  outside  all  an  asbestos  or  felt  jacket,  or 


Fig.  94. — Incubator. 

wooden  casing.  The  water  in  the  jacket  is  heated 
by  gas  and  maintained  at  some  constant  temperature 
by  a  thermo-regulator. 

Two  incubators  at  least  are  required  for  the  culti- 
vation of  bacteria  in  the  laboratory,  the  one  regulated 

174 


THERMOREGULATORS.  1 75 

to  maintain  a  temperature  of  37°  C.,  and  known  as 
the  "hot"  incubator;  the  other,  22°  C.,  and  known  as 
the  "cool"  or  "cold"  incubator. 

Thermo=regulators. — The  thermo-regulator  is  the 
most  essential  portion  of  the  incubator,  as  upon  its 
efficient  working  depends  the  maintenance  of  a  con- 
stant temperature  in  the  cultivation  chamber.  It  is 
also  used  in  the  fitting  up  of  water  and  paraffin  baths, 
and  for  many  other  purposes. 

Of  the  many  forms  and  varieties  of  thermo-regulator 
(other   than   electrical),  two  only  are   of   sufficiently 
general  use  to  need  mention.     In  one  of  these  the  flow 
of  gas  to  the  gas-jet  is  controlled  by 
the  expansion  or  contraction  of  mer- 
cury within  a  glass  bulb ;  in  the  other, 
by  alterations  in  the  position  of  the 
walls  of   a   hollow   metallic   capsule. 
They  are: 

(a)  Reichert's  (Fig.  95),  which  con- 
sists of  a  bulb   containing  mercury. 
Gas  enters  at  A,  and  passes  out  to 
the  jet  by  B.      As  the   temperature 
rises  the  mercury  expands  and  cuts 

rr     ^1  •  1          j_i          1  Fig.   95. — Reichert's 

off   the    mam    gas   supply,    thereby        fhermo-reguiator. 
reducing   the   temperature.      As  the 
temperature  falls  the  mercury  contracts  and  reopens 
the  narrow  tube  C.     By  means  of  a  thumbscrew  D 
(which  mechanically  raises  or  lowers  the  column   of 
mercury  irrespective  of  the  temperature)  and  the  aid 
of  a  thermometer  the  apparatus  can  be  set  to  keep  the 
incubator  at  any  desired  temperature.    With  this  form 
a  special  gas  burner  is  required,  with  separate  supply 
of  gas  to  a  pilot  jet  at  the  side. 

(b)  Capsule  regulator  consists  of  a  metal  capsule  filled 
with  a  liquid  which  boils  at  the  required  temperature, 
and  hermetically  sealed;  this  is  adjusted  in  the  interior 
of  the  incubator.     Soldered  to  the  upper  side  of  the 


1 76  INCUBATORS. 

capsule  is  a  thick  piece  of  metal  having  a  central  de- 
pression which  receives  the  lower  end  of  a  rigid  rod, 
by  means  of  which  the  movements  of  the  walls  of  the 
capsule  are  transmitted  to  the  gas  valve  fixed  outside 
the  incubator. 

Figure  96  represents  a  well-known  form  of  capsule 
regulator.  A  is  the  inlet  for  gas,  C  the  outlet  to 
burner  heating  the  water  jacket,  B  D  a  lever  pivoted 
to  standards  at  G,  and  acted  upon  by  the  capsule, 
through  the  needle  which  enters  the  socket  below 
the  screw  P. 

The  construction  of  the  valve  is  such  that,  when- 
ever the  end  B  of  the  lever  B  D  presses  on  the  disc 

below  the  end  B,  the 
main  supply  of  gas 
is  entirely  cut  off. 
At  such  times,  how- 
ever, a  very  small 
portion  of  gas 
passes  from  A  to  C, 
through  an  aperture 

Fig.  96.— Capsule  thermo- regulator.  inside  the  Valve,  the 

size  of  which  aper- 
ture can  be  adjusted  by  the  screw  needle  S,  hence  the 
gas  flame  below  the  incubator  is  never  extinguished. 

The  expansion  of  the  metal  walls  of  the  capsule, 
owing  to  the  boiling  of  its  contents,  provides  the 
motive  force  for  acting  upon  the  lever  B  D,  and  as  this 
expansion  only  takes  place  at  a  predetermined  tempera- 
ture, the  lever  will  only  be  acted  upon  when  the 
critical  temperature  is  reached,  no  sensible  effect  being 
produced  at  even  i°  C.  below  that  at  which  the  capsule 
is  desired  to  act. 

W  is  a  weight  sliding  on  the  lever  rod  D,  by  means 
of  which  the  boiling-point  of  the  liquid  in  the  capsule 
can  be  slightly  retarded,  and  a  range  of  about  two  de- 
grees obtained  with  any  particular  capsule. 


XIL  METHODS  OF  CULTIVATION. 

CULTIVATIONS  of  micro-organisms  are  usually  pre- 
pared in  the  laboratory  in  one  of  three  ways : 
Tube  cultures. 
Plate  cultures. 
Hanging=drop  cultures. 

These  may  be  incubated  either  aerobically  (i.  e., 
in  the  presence  of  oxygen)  or  anaerobically  (i.  e.,  in 
the  absence  of  oxygen,  or  in  the  presence  of  an  in- 
different gas,  such  as  hydrogen,  nitrogen,  or  carbon 
dioxide). 

With  regard  to  the  temperature  at  which  the  culti- 
vations are  grown,  it  may  be  stated  as  a  general  rule 
that  all  media  rendered  solid  by  the  addition  of  gela- 
tine are  incubated  at  20°  C.,  or  at  any  rate  at  a  tem- 
perature not  exceeding  22°  C.  (that  is,  in  the  "cold" 
incubator) ;  whilst  fluid  media  and  all  other  solid  media 
are  incubated  at  37°  C.  (that  is,  in  the  "hot"  incu- 
bator). Exceptions  to  this  rule  are  numerous.  For 
instance,  in  studying  the  growth  of  the  psychrophylic 
bacteria,  the  yeasts  and  the  moulds,  the  cold  incubator 
is  employed  for  all  media. 

Tube  cultivations  are  usually  packed  in  the  incubator 
in  small  tin  cylinders,  such  as  those  in  which  American 
cigarettes  are  sold.  Beakers  or  tumblers  may  be 
used  for  the  same  purpose,  but  are  not  so  convenient. 


AEROBIC 

The  Preparation  of  Tube  Cultivations. 

The  preparation  of  a  tube  cultivation  consists  in: 
(a)  Inoculating  a  tube  of  sterile  nutrient  medium 
with  a  portion  of  the  material  to  be  examined. 
12  177 


178  METHODS    OF   CULTIVATION. 

(6)  Incubating  the  inoculated  tube  at  a  suitable 
temperature. 

The  details  of  the  first  of  these  processes  must  be 
varied  somewhat  according  to  whether  the  tubes  of 
nutrient  media  are  inoculated  or  ''planted"  from— 

1.  Pre-existing  cultivations. 

2.  Previously  collected  morbid  material. 

3.  The  animal  body  direct. 

The  method  of  preparing  tube  cultivations  from 
pre-existing  cultivations  is  as  follows: 

1.  Fluid  Media  (e.  g.,  Nutrient  Bouillon). — 

i.  Flame  the  cotton- wool  plug  of  the  tube  contain- 


Fig.  97. — Inoculating  tubes,  seen  from  the  front. 

ing  the  cultivation  and  also  that  of  the  tube  of  sterile 
bouillon. 

2.  Hold  the  two  tubes,  side  by  side,  between  the 
left  thumb  and  the  first  and  third  fingers,  allowing 
the  sealed  ends  to  rest  on  the  dorsum  of  the  hand, 
and  separating  the  mouths  of  the  tubes   (which  are 
pointed  to  the  right)  by  the  tip  of  the  second  finger. 
Keep   the  tubes  as  nearly  horizontal  as   is   possible 
without  allowing  the  fluid  in  the  bouillon  tube  to  reach 
the  cotton- wool  plug. 

3.  Sterilise  the  platinum  loop  and  allow  it  to  cool.1 

4.  Grasp  the  plug  of  the  tube  containing  the  culti- 
vation between  the  little  finger  and  palm  of  the  hand 
and  remove  it  from  the  tube. 

1  See  also  method  of  opening  and  closing  culture  tubes,  page  69. 


SOLID    MEDIA. 


179 


5.  Grasp  the  plug  of  the  bouillon  tube  between  the 
fourth  finger  and  the  ball  of  the  thumb  and  remove 
it  from  the  tube. 

6.  Pass  the  platinum  loop  into  the  tube  containing 
the  culture, — do  not  allow  the  loop  to 

touch  the  sides  of  the  tube,  or  the 
handle  to  touch  the  medium, — and  re- 
move a  small  portion  of  the  growth; 
withdraw  the  loop  from  the  tube,  keep- 
ing the  infected  side  of  the  loop  down- 
wards. 

7.  Pass  the   loop   into   the   bouillon 
tube  almost  down  to  the  level  of  the 
fluid,  reverse  the  loop  so  that  the  in- 
fected side  faces  upwards,  emulsify  the 
portion  of  the  growth  in  the  moisture 
adhering  to  the  side  of  the  tube  which 
is  uppermost.     Withdraw  the  loop. 

8.  Replug  both  tubes. 

9.  Sterilise  the  platinum  loop. 

10.  Label  the  bouillon  tube  with  (a) 
the  name  of  the  organism  and  (b)  the 
date  of  inoculation. 

11.  Incubate. 

2.    Solid    Media. — Solid    media    are 
stored  in  tubes  in  one  of  two  ways : 

i.  Oblique  tube  or  slanted  tube  (Fig. 
98),  in  which  the  medium  has  been 
allowed  to  solidify  whilst  the  tube  was 
retained  in  an  inclined  position,  so  forming  an  exten- 
sive surface  of  medium  extending  from  the  bottom  of 
the  tube  almost  to  its  mouth. 

This  is  employed  for  "streak"  or  "smear"  cultiva- 
tions (Strichcultur) . 

NOTE. — Gelatine  and  agar  oblique  tubes  should  be 
freshly  "slanted"  before  use. 


Fig.    98. — Streak 
tube. 


i8o 


METHODS    OF    CULTIVATION. 


2.  Straight  tube  (Fig.  99),  in  which  the  medium 
forms  a  cylindrical  mass  in  the  lower  portion  of  the 
tube  and  presents  an  upper  surface  which  is  at  right 
angles  to  the  long  axis  of  the  tube. 

This  is  employed  for  "stab"  or  ''stick"  cultivations 
(Stichcultur\  or  by  inoculating    the   medium    whilst 
fluid,  and  allowing   to  solidify  in  this 
position,  for  "shake"  cultivations. 
Streak  Culture. — 

1.  Flame  the  plugs,  sterilise  the  plat- 
inum loop  (or  spatula).    Open  the  tubes 
and  charge  the  loop  as  in  previous  in- 
oculation. 

2.  Pass  the  infected  loop  to  the  bot- 
tom of  the  tube  to  be  inoculated  and 
draw  it,  as  lightly  as  possible,  along  the 
centre  of    the  surface  of  the  medium, 
terminating  the  "streak"  over  the  thin 
layer  of  medium  near  the  mouth  of  the 
tube. 

3.  Replug    the    tubes,    sterilise    the 
platinum  loop. 

4.  Label   the  newly  inoculated  tube 
and  incubate. 

Smear  Culture. — Proceed  generally  as 
in  streak  culture,  but  rub  the  infected 
loop  all  over  the  surface  of  the  medium, 
instead  of  restricting  the  inoculation  to 
a  narrow  line. 
Stab  Culture. — 
i.  Flame  the  plugs,  open  the  tubes, 
sterilise  the  platinum  needle  and  charge  it  with  the 
inoculum  as  in  the  previous  cultivations. 

2.  Pass  the  platinum  needle  into  the  tube  to  be 
inoculated  until  it  touches  the  centre  of  the  surface  of 
the  medium.  Now  thrust  it  deeply  into  the  sub- 
stance of  the  medium,  keeping  the  needle  as  nearly  as 


Fig.  99- — Straight 
tube. 


THE  PREPARATION  OF  PLATE  CULTURES.    l8l 


possible  in  the  axis  of  the  cylinder  of  medium.     Then 
withdraw  the  needle. 

3.  Replug  the  tubes.     Sterilise  the  platinum  needle. 

4.  Label  the  newly  planted  tube  and  incubate. 
Shake  Culture. — 

1.  Liquefy  a  tube  of  nutrient  gelatine  (or  agar,  or 
other  similar  medium),  by    heating  in  a  water-bath 
(Fig.  i  oo). 

2.  Inoculate    the   liquefied 
medium    and    label    it,    etc., 
precisely  as  if  dealing  with  a 
tube  of  bouillon. 

3.  Place  the  newly  planted 
tube  in  the  upright  position 
(e.  g.,  in  a  test-tube  rack)  and 
allow  it  to  solidify. 

4.  Label    the    tube;    when 
solid,  incubate. 


The  Preparation  of  Plate  Cul= 
tures. 

If  a  small  number  of  bac- 
teria are  suspended  in  lique- 
fied gelatine,  agar,  or  other 
similar  medium,  and  the  in- 
fected medium  spread  out  in 
an  even  layer  over  a  flat  sur- 
face and  allowed  to  solidify, 
each  individual  micro-organ- 
ism becomes  fixed  to  a  cer- 
tain spot  and  its  further  development  is  restricted  to 
tho  vicinity  of  this  spot.  After  a  variable  interval  the 
growth  of  this  organism  becomes  visible  to  the  naked 


Fig.  100. — Handy  form  of 
water-bath  for  melting  tubes  of 
agar  and  gelatine  previous  to 
slanting  them ;  or  to  making 
shake  cultures. 


eye  as  a  "colony."  This  is  the  principle  upon  which 
tho  method  of  plate  cultivation  is  based.  The  method 
itself  is  as  follows: 


182 


METHODS   OF   CULTIVATION. 


Apparatus  Required. — 

1.  Tripod  levelling  stand. 

2.  Large  shallow  glass  dish,  with  a  square  sheet  of  plate 

glass  to  cover  it. 

3.  Spirit  level. 

4.  Case  of  sterile  Petri  dishes. 

5.  Tubes  of  sterile  nutrient  media,  gelatine  (or  agar)  pre- 

viously liquefied  by  heating  in  the  water-bath  and 
cooled  to  42°  C.,  otherwise  the  heat  of  the  medium 
would  destroy  many,  if  not  all,  of  the  bacteria  intro- 
duced. 

6.  Tube  of  cultivation  to  be  planted  from. 

7.  Platinum  loop. 

8.  Bunsen  burner. 

9.  Grease  pencil. 


Fig.  101. — Plate-levelling  stand. 


Method  of  "Pouring"  Plates.— 

1.  Place  the  glass  dish  on  the  levelling  tripod  (Figs. 
10 1,  102),  fill  it  with  ice  water  if  gelatine  plates  are  to 
be  poured,  or  with  water   at  50°  C.  if   agar  is  to  be 
used;  cover  it  with  the  square  sheet  of  glass. 

2.  Place  the  spirit  level  on  the  sheet  of  glass  and  by 
means  of  the  levelling  screws  adjust  the  surface  of 
the  glass   to   the  horizontal. 

3.  Place  three  sterile  Petri  dishes  in  a  row  on  the 
surface  of  the  glass  plate  and  number  them  1,2,  and 
3,  from  left  to  right. 

4.  Number,  the  previously  liquefied  tubes  of  nutrient 
media   i,   2,   and  3.     Flame  the  plugs  and  see  that 
each  plug  can  be  readily  removed  from  the  mouth 
of  its  tube. 


METHOD    OF    "  POURING"    PLATES. 


5.  Add  one  loopful  of  the  inoculum  to  tube  No.  i, 
treating  the  liquefied  medium  as  bouillon.  After  re- 
plugging, grasp  the  tube  near  its  mouth  by  the  thumb 
and  first  finger  of  the  right  hand,  and  with  an  even 
circular  movement  of  the  whole  arm,  diffuse  the  inocu- 


Fig.  1 02. — Plate-levelling  stand,  side  view. 

lum  throughout  the  medium;  avoid  jerky  movements, 
as  these  cause  bubbles  of  air  to  form  in  the  medium. 

6.  Sterilise  the  platinum  loop,  and  add  two  loopfuls 
of  diluted  inoculum  to  tube  No.  2,  and  mix  as  before. 

7.  In   a   similar   manner  transfer  three  loopfuls  of 


Fig.  103. — Pouring  plates. 


and 


liquefied  medium  from  tube  No.  2  to  tube  No.  3, 
mix  thoroughly. 

8.  Flame  the  plug  of  tube  No.  i,  remove  it,  then 
flame  the  lips  of  the  tube;  slightly  raise  the  cover  of 
Petri  dish  No.  i,  introduce  the  mouth  of  the  tube; 
then,  elevating  the  bottom  of  the  tube,  pour  the  lique- 
fied medium  into  the  Petri  dish,  to  form  a  thin  layer. 


184  METHODS   OF   CULTIVATION. 

Remove  the  mouth  of  the  tube  and  close  the  "plate." 
If  the  medium  has  failed  to  flow  evenly  over  the  bottom 
of  the  plate,  raise  the  plate  from  the  levelling  platform 
and  by  tilting  in  different  directions  rectify  the  fault. 

9.  Pour  plates  No.  2  and  No.  3,  in  a  similar  manner, 
from  tubes  Nos.  2  and  3. 

10.  Label  the  plates  with  the  distinctive  name  or 
number  of  the  inoculum,  the  number  of  the  dilution, 
also  the  date. 

11.  Place  in  the  cool  incubator  for  three  or  more 
days,   as  may  be  necessary. 

In  this  way  colonies  may  be  obtained  quite  pure  and 
separate  from  each  other. 

In  plate  No.  i,  probably,  the  colonies  will  be  so 
numerous  and  crowded,  and  therefore  so  small,  as  to 
render  it  useless.  In  plate  No.  2  they  will  be  more 
widely  separated,  but  usually  No.  3  is  the  plate  reserved 
for  careful  examination,  as  in  this  the  colonies  are  usu- 
ally widely  separated,  few  in  number,  and  large  in  size. 

A  gar  plates  are  poured  in  a  similar  manner,  but  the 
agar  must  be  melted  in  boiling  water  and  then  allowed 
to  cool  to  45°  C.  or  42°  C.  in  a  carefully  regulated 
water-bath  before  being  inoculated,  and  the  entire  pro- 
cess must  be  carried  out  very  rapidly,  otherwise  the  agar 
will  have  solidified  before  the  operation  is  completed. 

NOTE. — In  pouring  plates,  tube  No.  i  (for  the  first 
dilution)  very  rarely  gives  a  plate  that  is  of  any  prac- 
tical value;  consequently  it  is  frequently  replaced  by 
a  tube  of  bouillon  or  sterile  salt  solution,  and  plate 
No.  i  is  not  poured. 

Hanging=drop  Cultivation. 

(a)  Fluid  Media.— 

i .  Prepare  first  and  second  dilutions  of  the  inoculum 
as  directed  for  plate  cultivations  (vide  page  182, 
sections  4  to  6),  substituting  tubes  of  nutrient  broth 
for  the  liquefied  gelatine. 


HANGING-DROP    CULTIVATION.  185 

2.  Sterilise  a  hanging-drop  slide  by  washing  thor- 
oughly in  water  and  drying,  then  plunging  it  into  a 
beaker  of  absolute  alcohol,   draining  off  the  greater 
part  of  the  spirit,  grasping  the  slide  in  a  pair  of  forceps, 
and  burning  off  the  remainder  of  the  alcohol  in  the 
flame. 

3.  Place  the  hanging-drop  slide  on  a  piece  of  blotting 
paper  moistened  with  2  per  cent,  lysol  solution  and 
cover  it  with  a  small  bell  glass  that  has  been  rinsed 
out  with  the  same  solution  and  not  dried. 

4.  Raise  the  bell  glass  slightly  and  smear  sterile 
vaseline  around  the  rim  of  the  metal  cell  by  means 
of  a  sterile  spatula  of  stout  platinum  wire. 

5.  Remove  a  clean  cover- slip  from  the  alcohol  pot 
with  sterile  forceps  and  burn  off  the  alcohol;  again 
raise  the  bell  glass  and  place  the  sterile  cover-slip  on 
the  blotting  paper  by  the  side  of  the  hanging-drop  slide. 

6.  Remove   a  drop  of  the  broth  from  the  second 
dilution  tube  with  a  large  platinum  loop;  raise  the 
bell  glass  and  deposit  the  drop  on  the  centre  of  the 
cover-slip.     Sterilise  the  loop. 

7.  Raise  the  bell  glass  sufficiently  to  allow  of  the 
cover-slip  being  grasped    with  forceps,  inverted,  and 
adjusted  over  the  cell  of  the  hanging-drop  slide.     Re- 
move the  bell  glass  altogether  and  press  the  cover-slip 
firmly  on  to  the  cell. 

8.  Either  incubate  and  examine  at  definite  intervals, 
or  observe  continuously  with  the  microscope,  using 
a  warm  stage  if  necessary  (Fig.  40) . 

(b)  Solid  Media. — Observing  precisely  similar  tech- 
nique, a  few  drops  of  liquefied  gelatine  or  agar  from 
the  second  dilution  tube  may  be  run  over  the  surface 
of  the  sterile  cover-slip  and  a  hanging-drop  plate  cul- 
tivation thereby  prepared. 

This  method  is  extremely  useful  in  connection  with 
the  study  of  yeasts,  and  in  this  connection  the  circular 
cell  on  the  hanging-drop  slide  is  replaced  by  a  rectangu- 


1 86  METHODS   OF   CULTIVATION. 

lar  cell  some  38  by  19  mm.,  and  the  gelatine  spread 
over  a  cover-slip  of  similar  size.  After  sealing  down 
the  preparation,  the  upper  surface  of  the  cover-slip 
may  be  ruled  into  squares  by  the  aid  of  the  grease 
pencil  or  a  writing  diamond. 


ANAEROBIC  CULTIVATIONS. 

Numerous  methods  have  been  devised  for  the  culti- 
vation of  anaerobic  bacteria,  the  majority  requiring 
the  employment  of  special  apparatus.  The  principle 
upon  which  any  method  is  based  and  upon  which  it 
depends  for  its  success  falls  under  one  or  another  of 
the  following  headings: 

(a)  Exclusion  of  air  from  the  cultivation. 

(b)  Exhaustion  of  air  from  the  vessel  containing  the 
cultivation  by  means  of  an  air  pump — i.  e.,  cultiva- 
tion in  vacua. 

(c)  Absorption  of  oxygen  from  the  air  in  contact 
with    the    cultivation    by    means    of    pyrogallic    acid 
rendered  alkaline  with  caustic  soda — i.  e.,  cultivation 
in  an  atmosphere  of  nitrogen. 

(d)  Displacement  of  air  by  an  indifferent  gas,  such 
as  hydrogen  or  coal  gas — i.  e.,  cultivation  in  an  atmos- 
phere of  hydrogen. 

(e)  A  combination  of  two  or  more  of  the  above 
methods. 

A  selection  of  the  simplest  and  most  generally  useful 
methods  is  given  here. 

Whenever  possible,  the  nutrient  media  that  are  em- 
ployed in  any  of  the  processes  should  contain  some 
easily  oxidisable  substance,  such  as  sodium  formate 
(0.4  per  cent.)  or  sodium  sulphindigotate  (o.i  per 
cent.),  which  will  absorb  all  the  available  oxygen  held 
in  solution  by  the  medium.  The  further  addition  of 
glucose,  2  per  cent.,  favors  the  growth  of  anaerobic 
bacteria  (vide  glucose  formate  bouillon,  page  142; 
sulphindigotate  bouillon,  page  143,  etc.). 


ANAEROBIC    CULTIVATIONS.  187 

Further,  it  is  advisable  to  seal  all  joints  between 
india-rubber  stoppers  and  tubulures  or  the  mouths 
of  the  tube  with  melted  paraffin. 

(A)  Method  I  (Hesse's  Method).— 

1.  Make  a  stab  culture  in  gelatine  or  agar,  choosing 
for  the  purpose   a  straight  tube   containing  a  deep 
column    of    medium,    and    thrusting    the    inoculating 
needle  to  the  bottom  of  the  tube. 

2.  Pour  a  layer  of  sterilised  oil  (olive  oil,  vaseline, 
or  petroleum),  i  or  2  cm.  deep,  upon  the  surface  of 
the  medium. 

3.  Incubate. 
Method  II.- 

1.  Make  an  Esmarch's  roll  cultivation  in  the  usual 
way. 

2.  Fill  the  lumen  of  the  tube  with  sterile  gelatine 
that  has  been  liquefied  by  heat  and  cooled  in  the 
water-bath  to  15°  C.     (At  this  temperature  the  gela- 
tine will  remain  fluid  for  only  a  few  minutes.) 

3.  Incubate. 

NOTE. — This  method  is  but  rarely  employed. 

Method  III. — This  method  is  only  available  when 
dealing  with  pure  cultivations. 

1.  Liquefy  a  tube  of  gelatine    (or  agar)   by  heat, 
pour  it  into  a  Petri  dish,  and  allow  it  to  solidify. 

2.  Inoculate  the  surface  of  the  medium  in  one  spot 
only. 

3.  Remove  a  cover-slip  from   the  pot   of   absolute 
alcohol  with  sterile  forceps;  burn  off  the  alcohol  in  the 
gas  flame. 

4.  Lower  the  now  sterile  cover-slip  carefully  on  to 
the  inoculated  surface  of  the  medium,   carefully  ex- 
cluding air  bubbles,  and  press  it  down  firmly  with  the 
points  of  the  forceps.     (A  sterile  disc  of  mica  may 
be  substituted  for  the  cover-slip.) 

5.  Incubate. 


i88 


METHODS    OF    CULTIVATION. 


Method  IV   (Roux's  Physical  Method).— 

1.  Prepare  tube  cultures  of  fluid  media   (or  solid 
media  rendered  fluid  by  heat)  in  the  usual  way. 

2.  Aspirate  some  of  the  inoculated  media  into  capil- 
lary pipettes. 

3.  Seal  both  ends  of  each  pipette  in  the  blowpipe 
flame. 

4.  Incubate. 

Method  V  (Roux's  Biological  Method).— 

1.  Plant  a  deep  stab,  as  in  method  I. 

2.  Pour  a  layer,  i  or  2  cm.  deep,  of  broth  cultiva- 
tion of   an  aerobe — e.  g.,  B.  aquatilis  sulcatus  or  B. 

prodigiosus — upon  the  surface  of  the 
medium;  or  an  equal  depth  of  lique- 
fied gelatine,  which  is  then  inoculated 
with  the  aerobic  organism. 

3.  Incubate. 

The  growth  of  the  aerobe  will  use 
up  all  the  oxygen  that  reaches  it  and 
will  not  allow  any  to  pass  through  to 
the  medium  below,  which  will  con- 
sequently remain  in  an  anaerobic 
condition. 

(B)  Method  VI.- 

1.  Prepare  tube  or  flask  cultiva- 
tions in  the  usual  way. 

2.  Replace    the   cotton- wool   plug 
by  an    india-rubber    stopper  perfor- 
ated with  one  hole  and  fitted  with  a 
length  of  glass  tubing  which  has  a 

constriction  about  3  cm.  above  the  stopper  and  is  then 
bent  at  right  angles  (Fig.  104).  The  stopper  and  glass 
tubing  are  sterilised  by  being  boiled  in  a  beaker  of 
water  for  five  minutes. 

3.  Connect  the  tube  leading  from  the  culture  vessel 
with  a  water  or  air  pump,  interposing  a  Wulff's  bottle 
fitted  as  a  wash-bottle  and  containing  sulphuric  acid. 


Fig.  104. — Vacuum 
culture. 


ANAEROBIC    CULTIVATIONS. 


189 


4.  Exhaust  the  air  from  the  culture  vessel. 

5.  Before  disconnecting  the  apparatus,  seal  the  glass 
tube  from  the  culture  vessel  at  the  constriction,  using 
the  blowpipe  flame. 

6.  Incubate. 

(C)  Method  VII  (Buchner's  Method).— 

Apparatus  and  Solutions  Required. — 

Buchner's  tube  (a  stout  glass  test-tube  23  cm.  long  and 
4  cm.  in  diameter,  fitted  with  india-rubber  stopper, 
Fig.  105). 

Ten  per  cent,  aqueous  solution  of  pyrogallic  acid. l 

Dekanormal  solution  of  caustic  soda. 

METHOD.— 

1.  Prepare  the  tube  cultivation  in  the  usual  way. 

2.  Moisten  the  india-rubber  stopper  of  the  Buchner's 
tube  with  water  and  see  that  it  fits  the 

mouth  of  the  tube  accurately. 

3.  Remove  the  stoppers  from  the  py- 
rogallic acid  and  caustic  soda  bottles. 

4.  Run  about   10  c.c.  of    the   pyro- 
gallic solution  into  the  Buchner's  tube 
(roughly,  use  5  c.c.  pyrogallic  solution 
for  every  100  c.c.  air  capacity  of  the 
receiving  vessel). 

5.  Add  about  i  c.c.  of  the  soda  solution. 

6.  Place  the   inoculated   tube  inside 
the  Buchner's  tube. 

7.  Fit  the  india-rubber  stopper  tightly 
into  the  mouth  of  the  Buchner's  tube. 


Fig.  105. — Buch- 
ner's tube. 


NOTE. — Sections  4  to  7  must  be  per- 
formed as  quickly  as  possible. 

8.  Restopper  the  pyrogallic  acid  and 
caustic  soda  bottles. 

9.  Place  Buchner's  tube  in  a  wire  support,  and  incu- 
bate. 

1  One  and  a  half  cubic  centimetres  of  hydrochloric  acid  should  be  added  to 
every  looo  c.c.  of  the  stock  pyrogallic  solution  to  prevent  oxidation. 


METHODS    OF   CULTIVATION. 

Method  VIII  (Wright's  Method).— 

1.  Prepare  tube  cultivation  in  the  usual  way. 

2.  Cut   off   that   portion   of   the   cotton- wool   plug 
projecting  above  the  mouth  of  the  tube  with  scissors, 
then  push  the  plug  into  the  tube  for  a  distance  of  2  or 
3  cm. 

3.  By  means  of  a  pipette  drop  about  i  c.c.  of  the 
pyrogallic  acid  solution  on  to  the  plug.     It  will  imme- 
diately be  absorbed  by  the  cotton- wool. 

4.  With  another  pipette  run  in  an  equal  quantity 
of  the  caustic  soda  solution. 

5.  Quickly  close  the  mouth  of  the  tube  with  a  tightly 
fitting  india-rubber  stopper. 

6.  Incubate. 

(D)  Method  IX.- 

Apparatus  Required. — 

Small  Ruffer's  or  Woodhead's  flask  (Fig.  23). 
Sterile  india-rubber  stopper. 
India-rubber  tubing. 
Glass  tubing. 
Metal  screw  clips. 

Cylinder  of  compressed   hydrogen   or   Kipp's   hydrogen 
apparatus. 

METHOD.— 

1.  Sterilise  a  glass  vessel,  shaped  as  in  a  Ruffer's  or 
Woodhead's  flask,  in  the  hot-air  oven.     (The  tubulure 
and    the    side    tubes   are  plugged  with  cotton- wool.) 
After  sterilisation,  fix  a  short  piece  of  rubber  tubing 
occluded  by  a  metal  clip  to  each  side  tube. 

2.  Inoculate  a  large  quantity  (e.  g.,  200  c.c.)  of  the 
medium.     Where  solid  media  are  employed  they  must 
first  be  liquefied  by  heat. 

3.  Remove  the  cotton- wool  plug  from  the  tubulure 
and  pour  the  inoculated  medium  into  the  glass  vessel. 

4.  Close  the  tubulure  by  means  of  an  india-rubber 
stopper  previously  sterilised  by  boiling  in  a  beaker 
of  water. 


ANAEROBIC   CULTIVATIONS. 


191 


5.  Connect  up  the  india-rubber  tubing  on  one  of 
the- side  tubes  with  a  cylinder  of  compressed  hydrogen 
(or  the  delivery  tube  of  a  Kipp's  hydrogen  apparatus, 
Fig.   1 06),  interposing  a  short  piece  of  glass  tubing; 
and   in   like    manner  connect  a  long  piece  of   rubber 
tubing  which  should  be  led  into  a  basin  of  water,  to 
the  opposite  side  tube. 

6.  Open  both  metal  clips  and  pass  hydrogen  through 
the  vessel  until  the  atmospheric  air  is  replaced  by 


Fig.  106. — Kipp's  hydrogen  apparatus  with  two  washing  bottles  containing 
lead  nitrate  and  silver  nitrate  solutions  respectively  to  remove  impurities. 


hydrogen.  This  is  determined  by  collecting  some  of 
the  gas  which  bubbles  through  the  water  in  the 
basin  in  a  test-tube  and  testing  it  by  means  of  a 
lighted  taper. 

7.  Close  the  metal  clip  on  the  tube  through  which 
the  gas  is  entering;  close  the  clip  on  the  exit  tube. 

8.  Disconnect  the  gas  apparatus. 

9.  Incubate. 


192 


METHODS   OF   CULTIVATION. 


Method  X  (Botkin's  Method).— 

Apparatus  Required. — 

Large  glass  dish  20  cm.  diameter  and  8  cm.  deep.  Flat 
leaden  cross  slightly  shorter  than  the  internal  diameter 
of  the  glass  dish. 

Bell  glass  about  15  cm.  diameter  and  20  to  25  cm.  high. 

Metal  frame  for  plate  cultivations. 

Or,  glass  battery  jar  for  tube  cultivations. 

Cylinder  of  compressed  hydrogen. 

Rubber  tubing. 

Two  pieces  of  U-shaped  glass  tubing  (each  arm  8  cm.  in 
length). 

Half  a  litre  of  glycerine. 

METHOD.— 

1.  Place  the  leaden  cross  inside  the  glass  dish,  rest- 
ing on  the  bottom. 

2.  Prepare  the  cultivations  in  the  usual  way. 

3.  Place  the  tube  cultivations  in  a  glass  battery  jar 

(or  the  plate  culti- 
vations on  a  metal 
frame),  resting  on 
the  centre  of  the 
leaden  cross. 

4.  Cover  the  cul- 
tivations   with   the 
bell  jar. 

5.  Adjust  the  (J- 
shaped     pieces     of 

•*-  glass  tubing  in  a 
vertical  position  on 
opposite  side  of  the 
bell  jar,  one  arm  of 
each  inside  the  jar, 
the  other  outside. 

Fig.  107.— Botkin's  apparatus.  Fix    a    short    length 

of     rubber     tubing 

clamped  with  a  metal  clip  to  each  of  the  outside  arms. 
6.  Fill  the  glass  dish  with  glycerine  to  a  depth  of 
about  5  cm.  (Fig.  107). 


ANAEROBIC   CULTIVATIONS. 


193 


7.  Connect  up  one  U-shaped  tube  with  the  hydrogen 
cylinder  by  means   of  rubber  tubing.     Replace   the 
atmospheric  air  by  hydrogen,  as  in  method  IX. 

8.  Clamp  the  tubes  and  disconnect  the  gas  apparatus. 

9.  Incubate. 

Method  XI  (Novy's  Method). - 

Apparatus  Required. — 

Jar  for  plate  cultivations  (Fig.  108). 

Or,  jar  for  tube  cultivations  (Fig.  109). 

Lubricant  for  stopper  of  jar  (beeswax  i  part,  olive  oil  4 

parts) . 

Rubber  tubing. 
Cylinder  of  compressed  hydrogen. 


Fig.  108. — Novy  jar  for 
plate  cultivations. 


Fig.  109. — Novy's  jar  for 
tube  cultivations. 


METHOD.— 

1.  Prepare  cultivations  in  the  usual  way. 

2.  Place  these  inside  the  jar. 

3.  Lubricate  the  stopper  and  insert  it  in  the  mouth 
of  the  jar,  with  the  handle  in  a  line  with  the  two  side 
tubes. 

4.  Connect  up  the  delivery  tube  a  with  the  hydrogen 
gas  supply  by  means  of  rubber  tubing. 

5.  Attach  a  piece  of  rubber  tubing  to  the  exit  tube 
b  and  collect  samples  of  the  issuing  gas  (over  water) 
and  test  from  time  to  time. 

13 


194  METHODS   OF   CULTIVATION. 

6.  When  the  air  is  completely  displaced  by  hydrogen, 
turn  the  handle  of  the  stopper  at  right  angles  to  the 
line  of  the  entry  and  exit  tubes;  this  seals  the  orifice 
of  both  tubes. 

7.  Disconnect  the  gas  apparatus  and  incubate. 
(E)  Method  XII  (Bullock's  Method).— 

Apparatus  Required. — 

Bullock's  jar. 

Pot  of  resin  ointment. 

Small  glass  dish  14  cm.  diameter  by  5  cm.  deep. 

Vessel  for  tube  cultures  or  metal  rack  for  plate  cultures. 

Pyrogallic  acid  powder. 

Cylinder  of  compressed  hydrogen. 

Geryk  or  other  air  pump. 

Rubber  tubing. 

Glass  tubing. 

Small  beaker  of  dekanormal  caustic  soda. 

Small  beaker  of  water. 

METHOD.— 

1.  Prepare  the  cultivations  in  the  usual  way. 

2.  Place  the  glass  dish  in  the  centre  of  the  glass 
slab,  and  stand  the  cultivations  inside  this. 

3.  Place  a  quantity  of  dry  pyrogallic  acid  in  a  heap 
at  one  side  of  the  glass  dish. 

4.  Smear  the  flange  of  the  bell  jar  with  resin  ointment 
and  apply  the  jar  firmly  to  the  glass  slab,  covering 
the  cultivations, — so  arranged  that  the  long  tube  passes 
with  its  lower  end  into  the  glass  dish  at  a  point  directly 
opposite  to  the  pyrogallic  acid  powder.     (This  is  to 
prevent  the  tube  getting  blocked  with  pyrogallic  acid 
during  the  next  step.)     Lubricate  the  two  stop-cocks 
with  resin  ointment. 

5.  Connect  up  the  short  tube  a  with  the  gas-supply 
by  means  of  rubber  tubing  and  open  both  stop-cocks. 

6.  When  the  air  is  displaced,  shut  off  the  stop-cock 
of  the  entry  tube,  then  that  of  the  exit  tube  b. 

7.  Connect  a  long,  straight  piece  of  glass  tubing  to 
the  long  tube  b  by  means  of  a  piece  of  rubber  tubing; 


ANAEROBIC    CULTIVATIONS. 


195 


and  connect  up  the  short  tube  a  to  the  air  pump  by 
means  of  pressure  tubing. 

8.  Open  the  stop-cock  of  tube  a  and  aspirate  a  small 
quantity,  say  100  c.c.,  of  gas  by  means  of  the  air  pump, 
so  creating  a  slight  vacuum.     Then  shut  off  the  stop- 
cock and  disconnect  the  air  pump. 

9.  Dip  the  long  glass  tube  (connected  with  6)  into 
the  beaker  of  soda  solution ;  open  the  stop-cock  and 
the   alkali  will  run 

down  the  long  tube 
and  come  into  con- 
tact with  the  dry 
pyrogallic  acid. 

10.  When  2  .or  3 
c.c.  of  soda  solution 
have    been   run   in, 
shut    off    the    stop- 
cock,    remove     the 
glass  tube  from  the 
soda    solution,    and 
place      it      in      the 
beaker  of  water. 

11.  In    a    similar 
manner  run  in  a  few 
cubic  centimetres  of 
water      and     again 

shut  the  stop-cock.  (This  serves  to  wash  out  the  tubes 
and  prevents  the  alkali  collecting  at  the  stop-cock  and 
exerting  a  corrosive  action  on  the  glass.) 

12.  Incubate. 

This  last  method  is  the  most  satisfactory  for  anae- 
robic cultivations,  as  by  its  means  complete  anaerobi- 
osis  can  be  obtained  with  the  least  expenditure  of  time 
and  trouble. 


Fig.  no. — Bullock's  jar. 


XHI.  METHODS  OF  ISOLATION. 

THE  work  in  the  preceding  sections,  arranged  to 
demonstrate  the  chief  biological  characters  of  bacteria 
in  general,  is  intended  to  be  carried  out  by  means  of 
pure  cultivations  of  various  organisms.  But  before 
undertaking  a  systematic  study  of  selected  bacteria, 
it  is  necessary  to  indicate  the  chief  methods  by  which 
one  or  more  organisms  may  be  isolated  in  a  state  of 
purity  from  a  mixture;  whether  that  mixture  exists 
as  an  impure  cultivation,  in  pus  and  other  morbid 
exudations,  infected  tissues,  or  water  or  food-stuffs. 


Fig.  III. — Haematocytometer  cell,  showing,  a,  section  through  the  centre  of 
the  cell,  and  b,  a  magnified  image  of  the  cell  rulings. 

Before  the  introduction  of  solid  media  the  only 
method  of  obtaining  pure  cultivations  was  by  "dilu- 
tion"— by  no  means  a  reliable  method.  "  Dilution" 
consisted  in  estimating  approximately  the  number  of 
bacteria  present  in  a  given  volume  of  fluid  (by  means 
of  a  graduated-celled  slide  resembling  a  haemato- 
cytometer,  Fig.  in.),  diluting  the  fluid  by  the  addi- 
tion of  sterile  water  or  bouillon  until  a  given  volume 
(usually  i  c.c.)  of  the  dilution  should  contain  but  one 
organism.  By  planting  this  volume  of  the  fluid  into 
several  tubes  or  flasks  of  nutrient  media,  it  occasion- 

196 


PLATE    CULTIVATIONS.  197 

ally  happened  that  the  resulting  growth  was  the  pro- 
duct of  one  individual  microbe.  A  method  so  uncer- 
tain is  now  fortunately  replaced  by  many  others, 
both  reliable  and  convenient,  and  in  those  methods 
selected  for  description  here,  the  isolation  of  the  re- 
quired bacteria  is  effected— 

1.  By  plate  cultivation: 

(a)  Gelatine. 

(b)  Agar. 

(c)  Serum  agar. 

(d)  Blood  agar. 

2.  By  Bsmarch's  roll  cultivation: 

(a)  Gelatine. 

(b)  Agar. 

3.  By  serial  cultivation. 

4.  By  differential  media. 

(a)  Selective. 

(b)  Deterrent. 

5.  By  differential  incubation. 

6.  By  differential  sterilisation. 

7.  By  differential  atmosphere  cultivation. 

8.  By  animal  inoculation. 

The  selection  of  the  method  to  be  employed  in  any 
specific  instance  will  depend  upon  a  variety  of  cir- 
cumstances, and  often  a  combination  of  two  or  more 
will  ensure  a  quicker  and  more  reliable  result  than 
a  rigid  adherence  to  any  one  method.  Experience  is 
the  only  reliable  guide,  but  as  a  general  rule  the  use 
of  either  the  first  or  the  third  method  will  be  found 
most  convenient,  affording  as  they  do  an  opportunity 
for  the  simultaneous  isolation  of  more  than  one  of  the 
bacteria  present  in  a  mixture. 

1.  Plate  Cultivations.— 

(a)  Gelatine  (vide  page  145). 

(b)  Agar  (vide  page  149). 

(c)  Alkaline  serum  agar  (vide  page  157). 

These   plates   are   poured    in   a    manner    precisely 


198  METHODS   OF   ISOLATION. 

similar  to  that  adopted  for  nutrient  gelatine  and  nu- 
trient agar  plates   (vide  page  182). 
(c')  Serum  Agar.— 

1.  Melt  three  tubes  of  nutrient  agar,  label  them  1,2, 
and   3,   and  place  them,   with  three  tubes   of  sterile 
fluid  serum,  also  labelled  1,2,  and  3,  in  a  water-bath 
regulated  at  45°  C. ;  allow  sufficient  time  to  elapse  for 
the  temperature  of  the  contents  of  each  tube  to  reach 
that  of  the  water-bath. 

2.  Make  three  dilutions  of  the  inoculum  in  the  three 
liquid  serum  tubes,  treating  them  exactly  as  if  they 
were  tubes  of  liquefied  gelatine  (vide  page  183) ;  replace 
them  in  the  water-bath. 

3.  Take  serum  tube  No.    i   and  agar  tube  No.    i. 
Flame  the  plugs  and  remove  them  from  the  tubes 
(retaining  the  plug  of  the  agar  tube  in  the  hand); 
flame  the  mouths  of  the  tubes,  pour  the  serum  into 
the  tube  of  liquefied  agar  and  replace  the  plug  of  the 
agar  tube. 

4.  Mix  thoroughly  and  pour  plate  No.   i  secundum 
artem. 

5.  Treat  the  remaining  dilutions  in  a  similar  fashion, 
and  pour  plates  Nos.  2  and  3  in  the  usual  way. 

6.  Label  and  incubate. 
(d)  Human  Blood  Agar. — 

1.  Melt  a  tube  of  sterile  agar  and  pour  it  into  a 
sterile  plate;  let  it  set. 

2.  Collect  a  few  drops  of  human  blood,  under  all 
aseptic  conditions,  in  a  sterile  capillary  pipette. 

3.  Raise  the  cover  of  the  Petri  dish  very  slightly, 
insert  the  extremity  of  the  capillary  pipette,  and  de- 
posit the  blood  on  the  centre  of  the  agar  surface. 
Close  the  dish. 

4.  Charge  a  platinum  loop  (or  a  sterilised  camel's 
hair  brush)  with  a  small  quantity  of  the  inoculum. 
Raise  the  cover  of  the  plate,  introduce  the  loop,  mix 


ESMARCH'S  ROLL  CULTIVATION.  199 

its  contents  with  the  drop  of  blood,  and  finally  smear 
the  mixture  over  the  surface  of  the  agar. 

5.  Withdraw  the  loop  and  close  the  plate. 

6.  Label  and  incubate. 

(If  considered  necessary,  two,  three,  or  more  similar 
plates  may  be  inoculated  in  series.) 
2.  Esmarch's  Roll  Cultivation.- 
(a)  Gelatine.— 

1.  Liquefy  three  tubes  of  gelatine  by  heat. 

2.  Prepare  three  dilutions  of  the  inoculum   (as  de- 
scribed for  plate  cultivations). 

3.  Roll  the   tubes,   held    almost  horizontally,   in    a 
groove  made  in  a  block  of  ice,  until  the  gelatine  has 


Fig.  112. — Esmarch's  roll  culture  on  block  of  ice. 

set  in  a  thin  film  on  the  inner  surface  of  tube  (Fig.  112); 
or  under  the  cold-water  tap. 

(6)  Agar  roll  cultures  are  made  in  precisely  the  same 
way  as  gelatine  roll  cultures,  but  in  order  that  the 
medium  may  adhere  firmly  to  the  glass,  the  agar  used 
for  the  purpose  should  have  i  per  cent,  gelatine  or  i 
per  cent,  gum  arabic  added  to  it  before  sterilisation. 

Roll  cultivations,  which  served  a  most  important 
purpose  in  the  days  before  the  introduction  of  Petri 
dishes  for  plate  cultivations,  are  now  seldom  prepared, 
and  are  in  point  of  fact  practically  obsolete. 

3.  Serial    Cultivations. — These     are    usually    made 


200  METHODS   OF   ISOLATION. 

upon  agar  or  blood-serum,  although  gelatine  may  also 
be  used.     The  method  is  as  follows: 

1.  Take  at  least  six  "slanted"  tubes  of  media  and 
number  them  consecutively. 

2.  Flame  all  the  plugs  and  see  that  each  can  be 
readily  removed. 

3.  Charge  the  platinum  loop  with  a  small  quantity 
of  the  inoculum,  observing  the  usual  routine,  and  plant 
tube  No.  i,  smearing  thoroughly  all  over  the  surface. 
If  any  water  of  condensation   has   collected   at  the 
bottom  of  the  tube,  use  this  as  a  diluent  before  smear- 
ing the  contents  of  the  loop  over  the  surface  of  the 
medium. 

4.  Without  sterilising  or  recharging  the  loop,  inocu- 
late tube  No.  2. 

5.  In  like  manner  plant  the  remainder  of  the  tubes 
in  the  series. 

6.  Label    with    distinctive   name    or   number,    and 
date;  incubate. 

The  growth  that  ensues  in  the  first  two  or  three 
tubes  of  the  series  will  probably  be  so  crowded  as  to  be 
useless.  Towards  the  end  of  the  series,  however,  dis- 
crete colonies  will  be  found,  each  of  which  can  be 
transferred  to  a  fresh  tube  of  nutrient  medium  without 
risk  of  contamination  from  the  neighboring  colonies. 

4.  Differential  Media.— 

(a)  Selective. — Some  varieties  of  media  are  specially 
suitable  for  certain  species  of  bacteria  and  enable  them 
to  overgrow  and  finally  choke  out  other  varieties;  e.g., 
wort  is  the  most  suitable  medium-base  for  the  growth 
of  torulae  and  yeasts  and  must  always  be  employed 
when  pouring  plates  for  the  isolation  of  these  organisms. 
To  obtain  a  pure  cultivation  of  yeast  from  a  mixture 
containing  bacteria  as  well,  it  is  sufficient  to  inoculate 
wort  from  the  mixture  and  incubate  at  37°  C.  for 
twenty-four  hours.  Plant  a  fresh  tube  of  wort  from 
the  resulting  growth  and  incubate.  Repeat  the  pro- 


DIFFERENTIAL   STERILISATION.  2OI 

cess  once  more,  and  from  the  growth  in  this  third 
tube  plant  a  streak  on  wort  gelatine,  and  incubate 
at  20°  C.  The  resulting  growth  will  almost  certainly 
be  a  pure  culture  of  the  yeast. 

(b)  Deterrent. — The  converse  of  the  above  also 
obtains.  Certain  media  possess  the  power  of  inhibiting 
the  growth  of  a  greater  or  less  number  of  species. 
For  instance,  media  containing  carbolic  acid  to  the 
amount  of  i  per  cent,  will  inhibit  the  growth  of  prac- 
tically everything  but  the  Bacillus  coli  communis. 

5.  Differential  Incubation. — In  isolating  certain  bac- 
teria,  advantage  is  taken  of  the  fact  that  different 
species  vary  in  their  optimum  temperature.     A  mix- 
ture containing  the  Bacillus  typhosus  and  the  Bacillus 
aquatilis  sulcatus,  for  example,  may  be  planted  on 
two  slanted  agar  tubes,  the  one  incubated  at  40°  C., 
and  the  other  at    12°  C.     After  twenty-four  hours' 
incubation  the  first  will  show  a  pure  cultivation  of  the 
Bacillus  typhosus,  whilst  the  second  will  be  an  almost 
pure  culture  of  the  Bacillus  aquatilis. 

6.  Differential  Sterilisation.— 

(a)  Non-sporing  Bacteria. — Similarly,  advantage  may 
be  taken  of  the  varying  thermal  death-points  of  bac- 
teria.    From  a  mixture  of  two  organisms  whose  ther- 
mal death-points  differ  by,  say,  4°  C. — e.  g.,  Bacillus 
pyocyaneus,  thermal  death-point  55°  C.,  and  Bacillus 
mesentericus  vulgatus,  thermal  death-point  60°  C. — a 
pure  cultivation  of  the  latter  may  be  obtained  by  heat- 
ing the  mixture  in  a  water-bath  to  58°  C.  and  keeping 
it  at  that  point  for  ten  minutes.     The  mixture  is  then 
planted  on  to  fresh  media  and  incubated,  when  the  re- 
sulting growth  will  be  found  to  consist  entirely  of  the 
B.  mesentericus. 

(b)  Sparing  Bacteria. — This  method  is  found  to  be 
of  even  greater  practical  value  when  applied  to  the 
differentiation  of  a  spore-bearing  organism  from  one 
which  does  not  form  spores.     In  this  case  the  mixture 


202 


METHODS    OF    ISOLATION. 


is  heated  in  a  water-bath  at  80°  C.  for  fifteen  to  twenty 
minutes.  At  the  end  of  this  time  the  non-sporing 
bacteria  are  dead,  and  cultivations  made  from  the 
mixture  will  only  yield  a  growth  resulting  from  the 
germination  of  the  spores  only. 

Differential  sterilisation  at  80°  C.  is  most  conveni- 
ently carried  out  in  a  water-bath  of  special  construc- 
tion, designed  by  Balfour  Stewart  (Fig.  113).  It  con- 
sists of  a  double- walled  copper  vessel 
mounted  on  legs,  and  provided  with 
a  tubulure  communicating  with  the 
space  between  the  walls.  This  space 
is  nearly  filled  with  benzole  (boiling- 
point  80°  C.),  and  to  the  tubulure  is 
fitted  a  long  glass  tube,  some  2  metres 
long  and  about  0.75  cm.  diameter, 
serving  as  a  condensing  tube.  The 
interior  of  the  vessel  is  partly  filled 
with  water  and  covered  with  a  lid 
which  is  perforated  for  a  thermom- 
eter. This  latter  dips  into  the  water 
and  records  its  temperature.  A  very 
small  Bunsen  flame  under  the  appa- 
ratus suffices  to  keep  the  benzole 
boiling  and  the  water  within  at  a 
\  constant  temperature  of  80°  C.  The 
bath  is  thus  always  ready  for  use. 

Fig.  113. — Benzole  ,,  /^  ... 

bath.  METHOD. — To  use  the  apparatus, 

i.  Place  some  of  the  mixture  it- 
self, if  fluid,  containing  the  spores,  or  an  emulsion  of 
the  same  if  derived  from  solid  material,  in  a  test-tube. 

2.  Immerse  the  test-tube  in  the  water  contained 
in  the  benzole  bath,  taking  care  that  the  upper  level 
of  the  liquid  in   the  tube   is   at  least  2    cm.  beneath 
the  surface  of  the  water  in  the  copper  vessel. 

3.  The  temperature  of  the  water,  of  course,  falls  a 
few  degrees  after  opening  the  bath  and  introducing 


DIFFERENTIAL   ATMOSPHERE    CULTIVATION.       203 

a  tube  of  colder  liquid,  but  after  a  few  minutes  the 
temperature  will  have  again  reached  80°  C. 

4.  When    the    thermometer   again   records    80°   C., 
note  the  time,  and  fifteen  minutes  later  remove  the 
tube  containing  the  mixture  from  the  bath. 

5.  Make  cultures  upon  suitable  media;  incubate. 

7.  Differential  Atmosphere  Cultivation.— 

(a)  By  adapting  the  atmospheric  conditions  to  the 
particular  organism  it  is  desired  to  isolate,  it  is  com- 
paratively easy  to   separate  a  strict   aerobe  from  a 
strict    anaerobe,    and   vice   -versa.     In    the   first   case, 
however,  it  is  important  that  the  cultivations  should 
be  made  upon  solid  media,  for  if  carried  out  in  fluid 
media  the  aerobes  multiplying  in  the  upper  layers  of 
fluid   render   the   depths    completely   anaerobic,    and 
under  these  conditions  the  growth  of  the  anaerobes  will 
continue  unchecked. 

(b)  When   it   is   desired   to   separate   a  facultative 
anaerobe  from  a  strict  anaerobe,  it  is  generally  suffi- 
cient to  plant  the  mixture  upon  the  sloped  surface 
agar,    incubate   aerobically   at   37°   C.,    and   examine 
carefully  at  frequent  intervals.     At  the  first  sign  of 
growth,  subcultivations  must  be  prepared  and  treated 
in  a  similar  manner.     As  a  result  of  these  rapid  sub- 
cultures, the  facultative  anaerobe  will  be  secured  in 
pure  culture  at  about  the  third  or  fourth  generation. 

(c)  If,  on  the  other  hand,  the  strict  anaerobe  is  the 
organism  required  from  a  mixture  of  facultative  and 
strict  anaerobes,  pour  plates  of  glucose  formate  agar 
(or  gelatine)  in  the  usual  manner,  place  them  in  a 
Bullock's  or  Novy's  jar,  and  incubate  at  a  suitable 
temperature.     Pick   off  the   colonies   of   the  required 
organism  when  the  growth  appears,  and  transfer  to 
tubes  of  the  various  media. 

Incubate  under  suitable  conditions  as  to  tempera- 
ture. 

8.  Animal  Inoculation. — Finally,  when  dealing  with 


204  METHODS   OF   ISOLATION. 

pathogenic  organisms,  it  is  often  advisable  to  inoculate 
some  of  the  impure  culture  (or  even  some  of  the  original 
materies  morbi)  into  an  animal  specially  chosen  on  ac- 
count of  its  susceptibility  to  the  particular  pathogenic 
organism  it  is  desired  to  inoculate.  Indeed,  with  some 
of  the  more  sensitive  and  strictly  parasitic  bacteria  this 
method  of  animal  inoculation  is  practically  the  only 
method  that  will  yield  a  satisfactory  result. 


XIV.  METHODS  OF  IDENTIFICATION. 

IN  order  to  identify  an  organism  after  nutrient 
media  have  been  inoculated,  and  tube,  plate,  and 
other  cultivations  prepared,  these  are  incubated  under 
suitable  conditions  as  to  temperature  and  environ- 
ment, are  examined  from  time  to  time  (a)  macroscopi= 
cally,  (b)  by  microscopical  methods,  (c)  by  chemical 
methods,  (d)  by  physical  methods,  (e)  by  inoculation 
methods,  and  the  results  of  these  examinations  duly 
recorded. 

It  must  be  stated  definitely  that  no  micro-organism 
can  be  identified  by  any  one  character  or  property, 
whether  microscopical,  biological,  or  chemical,  but 
that  on  the  contrary  its  entire  life  history  must  be 
carefully  studied  and  then  its  identity  established 
from  a  consideration  of  the  sum  total  of  these  observa- 
tions. 

In  order  to  give  to  the  recorded  results  their  maxi- 
mum value  it  is  essential  that  they  should  be  exact 
and  systematical,  therefore  some  such  scheme  as  the 
following  should  be  adhered  to ;  and  especially  is  this 
necessary  in  describing  an  organism  not  previously 
isolated  and  studied. 

SCHEME  OF  STUDY. 

Designation : 

Originally  isolated  by in  1 8 . . . ,  from 


.  Cultural   Characters. — (Vide  Macroscopical   Exam- 
ination of  Cultivation,  page  207.) 
Gelatine  plates, 

Gelatine  streak, 

r\  1  .•        .  ••  at  20   C. 

Gelatine  stab, 

Gelatine  shake, 

205 


206  METHODS    OF    IDENTIFICATION. 


Agar  plates, 

Agar  streak  or  smear, 

Agar  stab, 


Inspissated  blood-serum, 


at  20°  C.  and  37°  C. 


Bouillon, 
Litmus  milk, 
Potato, 

Special  media  for  the  purpose  of  demonstrating 
characteristic  reactions. 

2.  Morphology. — (Vide   Microscopical  Examination  of 

Cultivations,  page  218.) 
Vegetative  forms: 

Shape. 

Size. 

Motility. 

Flagella  (if  present). 

Capsule  (if  present). 

Involution  forms. 

Pleomorphism   (if  observed). 
Sporing  forms  (if  observed).     Of  which  class? 
Staining  reactions. 

3.  Biology. — (Vide  Physical  Examination  of  Cultures, 

page  238.) 
Vitality. 

Resistance  to  lethal  agents: 
Physical : 
Light. 
Colours. 

Chemical  germicides. 
Atmosphere. 
Temperature. 

Reaction  of  nutrient  media. 
Agglutination  reaction. 

4.  Chemical  Products  of  Growth. — (Vide  Chemical  Ex- 

amination of  Cultivations,  page  221.) 
Chromogenesis. 
Photogenesis. 


MACROSCOPICAI,   EXAMINATION.  207 

Enzyme  formation. 

Fermentation  of  carbohydrates: 

In  glucose  gelatine  shake  cultivation. 
In  saccharose  gelatine  or  bouillon. 
In  lactose  gelatine  or  bouillon. 
In  maltose  gelatine  or  bouillon. 
In  glycerine  bouillon  or  bouillon. 
Acid  formation. 
Alkali  formation  (if  present). 
Indol  formation. 
Phenol  formation. 
Reducing  and  oxidising  agents. 
Gas  formation. 
5.  Pathogenicity: 

Susceptible  animals. 

Immune   animals. 

Experimental  inoculation,  symptoms  of  disease. 

Post-mortem   appearances. 

Virulence : 

Length  of  time  maintained. 
Upon  what  medium? 
Minimal  lethal  dose. 

Is  virulence  readily  exalted  and  attenuated  ? 
Toxin  formation. 

MACROSCOPICAL  EXAMINATION  OF  CULTIVATIONS. 

In  describing  the  naked-eye  and  low-power  appear- 
ances of  the  bacterial  growth  the  descriptive  terms 
introduced  by  Chester  (and  included  in  the  following 
scheme)  should  be  employed. 

Solid    Media. 

Plate   Cultures. — 

Gelatine. — Note  the  presence  or  absence  of  lique- 
faction of  the  surrounding  medium.  If  liquefaction 
is  present,  note  shape  and  character  (vide  page  215, 
''stab"  cultures). 


208  METHODS   OF   IDENTIFICATION. 

A  gar. — No  liquefaction  takes  place  in  this  medium. 
The  liquid  found  on  the  surface  of  the  agar  (or  at  the 
bottom  of  the  tube  in  agar  tube  cultures)  is  merely 
water  which  has  been  expressed  during  solidification 
and  has  subsequently  condensed. 

Gelatine  and  Agar. — Examine  the  colonies  at  various 
intervals — 

(a)  With  the  naked  eye. 

(b)  Under  a  low  power  (i  inch)  of  the  microscope, 
or  by  means  of  a  small  dissecting  microscope. 

Distinguish  superficial  from  deep  colonies  and  note 
the  characters  of  the  individual  colonies. 


a  b  c 

Fig.  114. — Types  of  colonies:  a,  Cochleate  ;  3,  amoeboid;  c>  mycelioid. 

(A)  Size. — The  diameter  in  millimetres,  at  the  various 
ages. 

(B)  Shape.- 

Punctiform :  Dimensions  too  slight  for  defining  form 
by  naked  eye;  minute,  raised,  hemispherical. 

Round:  Of  a  more  or  less  circular  outline. 

Elliptical:  Of  a  more  or  less  oval  outline. 

Irregular. 

Fusiform:  Spindle-shaped,  tapering  at  each  end. 

Cochleate :  Spiral  or  twisted  like  a  snail  shell  (Fig. 
1 14,  a). 


MACROSCOPICAL  EXAMINATION.  2Og 

Amoeboid:  Very  irregular,  streaming  (Fig.  114,  b). 

Mycelioid:  A  filamentous  colony,  with  the  radiate 
character  of  a  mould  (Fig.  114,  c). 

Filamentous:  An  irregular  mass  of  loosely  woven 
filaments  (Fig.  115,  a). 

Floccose:  Of  a  dense  woolly  structure. 

Rhizoid:  Of  an  irregular,  branched,  root-like  char- 
acter (Fig.  115,  6). 

Conglomerate:  An  aggregate  of  colonies  of  similar 
size  and  form  (Fig.  115,  c). 


a  b  c  d 

Fig.  115. — Types  of  colonies:  a,  Filamentous;  b,  rhizoid ;  <:,  conglomerate; 

d,  toruloid. 

Toruloid:  An  aggregate  of  colonies,  like  the  budding 
of  the  yeast  plant  (Fig.  115,  d). 

Rosulate:  Shaped  like  a  rosette. 

(C)  Surface  Elevation. — 

i.  General  Character  of  Surface  as  a  Whole: 

Flat:  Thin,  leafy,  spreading  over  the  surface  (Fig. 
116,  a). 

Effused:  Spread  over  the  surface  as  a  thin,  veily 
layer,  more  delicate  than  the  preceding. 

Raised:  Growth  thick,  with  abrupt  terraced  edges 
(Fig.  1 1 6,  b). 

Convex:  Surface  the  segment  of  a  circle,  but  very 
flatly  convex  (Fig.  116,  c). 
14 


210 


METHODS    OF    IDENTIFICATION. 


Pulvinate:  Surface  the  segment  of  a  circle,  but  de- 
cidedly convex  (Fig.  116,  d). 

Capitate :  Surface  hemispherical 
(Fig.  1 1 6,  e). 

Umbilicate:  Having  a  central  pit 
or  depression  (Fig.  116,  /). 

Umbonate:  Having  a  central 
convex  nipple-like  elevation  (Fig. 
116,  g). 

2.  Detailed  Characters  of  Surface: 
Smooth:    Surface    even,    without 
any  of  the  following  distinctive  char- 
acters. 

Alveolate:  Marked  by  depressions 
separated  by  thin  walls  so  as  to  re- 
semble a  honeycomb  (Fig.  117). 

Punctate:  Dotted  with  punctures 
like  pin-pricks. 

Bullate:  Like  a  blistered  surface, 
rising  in  convex  prominences,  rather 
coarse. 

Vesicular:  More  or  less  covered 
with  minute  vesicles  due  to  gas 
formation;  more  minute  than  bul- 
late. 

Verrucose :  Wart-like,  bearing  wart-like  prominences. 
Squamose:  Scaly,  covered 
with  scales. 

Echinate :  Beset  with 
pointed  prominences. 

Papillate:  Beset  with  nip- 
ple or  mamma-like  pro- 
cesses. 

Rugose :  Short  irregular 
folds,  due  to  shrinkage  of 
surface  growth. 

Corrugated:  In. long  folds,  due  to  shrinkage. 


Fig.  1 1 6. — Surface 
elevation  of  colonies : 
a,  Flat ;  b,  raised ; 
r,  convex  ;  d,  pulvin- 
ate  ;  ^,  capitate  ;  f, 
umbilicate ;  g,  um- 
bonate. 


Fig.  117. — Types  of  colonies — 
alveolate. 


MACROSCOPICAL   EXAMINATION. 


211 


Contoured :  An  irregular  but  smoothly  undulating 
surface,  like  the  surface  of  a  relief  map. 

Rimose:  Abounding  in  chinks,  clefts,  or  cracks. 

(D)  Internal  Structure  of  Colony  (Microscopical). — 

Refraction  Weak:  Outline  and  surface  of  relief  not 
strongly  denned. 

Refraction  Strong:  Outline  and  surface  of  relief 
strongly  defined;  dense,  not  filamentous  colonies. 

i.  General: 

Amorphous:  Without  definite  structure,  as  below 
specified. 


a  b  c 

Fig.  1 1 8. — Types  of  colonies:  a,  Grumose  ;  £,  moruloid  ;  cy  clouded. 

Hyaline:  Clear  and  colourless. 

Homogeneous:  Structure  uniform  throughout  all 
parts  of  the  colony. 

Homochromous :  Colour   uniform  throughout. 

2.  Granulations  or  Blotchings: 

Finely  granular. 

Coarsely  granular. 

Grumose:  Coarser  than  the  preceding,  with  a 
clotted  appearance,  and  particles  in  clustered  grains 
(Fig.  1 1 8,  a). 

Moruloid :  Having  the  character  of  a  mulberry,  seg- 


212 


METHODS   OF   IDENTIFICATION. 


mented,  by  which  the  colony  is  divided  in  more  or 
less  regular  segments  (Fig.  118,  b). 

Clouded:  Having  a  pale  ground,  with  ill-defined 
patches  of  a  deeper  tint  (Fig.  118,  c). 

3.  Colony  Marking  or  Striping: 

Reticulate:  In  the  form  of  a  network,  like  the  veins 
of  a  leaf  (Fig.  119,  a). 


a  .        o  c 

Fig.  119. — Types  of  colonies  :  «,  Reticulate;  bt  gyrose  ;  c,  marmorated. 


Areolate:  Divided  into  rather  irregular,  or  angular, 
spaces  by  more  or  less  definite  boundaries. 

Gyrose:  Marked  by  wavy  lines,  indefinitely  placed 
(Fig.  119,  b). 

Marmorated :  Showing  faint,  irregular  stripes,  or  tra- 
versed by  vein-like  markings,  as  in 
marble  (Fig.  119,  c). 

Rivulose :  Marked  by  lines  like  the 
rivers  of  a  map. 

Rimose:    Showing   chinks,  cracks, 
or  clefts. 

4.  Filamentous  Colonies: 
Filamentous:   As   already  defined. 
Floccose:   Composed  of  filaments, 
densely  placed. 

Curled:  Filaments  in  parallel  strands,  like  locks  or 
ringlets,  as  in  agar  colonies  of  B.  anthracis. 


Fig.   1 20. — Types  of 
colonies — curled. 


MACROSCOPICAL   EXAMINATION.  213 

(E)  Edges  of  Colonies. — 

Entire :  Without  toothing  or  division  (Fig.  121,  a) . 

Undulate:  Wavy  (Fig.  121,  b). 

Repand :  Like  the  border  of  an  open  umbrella  (Fig. 

121,  C). 

Erose:     As    if    gnawed,    irregularly    toothed    (Fig. 
121,  d). 


Fig.   121. — Edges  of  colonies:  a,  Entire;  b,  undulate;  c,  repand  ;  d,  erose. 

Lobate. 

Lobulate:  Minutely  lobate  (Fig.  122,  e). 
Auriculate:  With  ear-like  lobes  (Fig.  122,  /). 
Lacerate:  Irregularly  cleft,  as  if  torn  (Fig.  122,  g). 
Fimbriate:  Fringed  (Fig.  122,  h}. 
Ciliate:  Hair-like  extensions,  radiately  placed  (Fig. 
122,7). 
Tufted. 

Filamentous:  As  already  defined. 
Curled:  As  already  defined. 


Fig.  122. — Edges  of  colonies  :  <?,  Lobar-lobulate  ;  /,  auriculate  ;  g,  lacerate  ; 
^,  fimbriate  ;  /,  ciliate. 


(F)  Optical  Characters  (after  Shuttleworth).— 
i.  General  Characters: 
Transparent:  Transmitting  light. 
Vitreous:  Transparent  and  colourless. 


214  METHODS    OF   IDENTIFICATION. 

Oleaginous:  Transparent  and  yellow;  olive  to  lin- 
seed-oil coloured. 

Resinous:  Transparent  and  brown,  varnish  or  resin- 
coloured. 

Translucent:  Faintly  transparent. 

Porcelaneous :  Translucent  and  white. 

Opalescent:  Translucent;  greyish- white  by  reflected 
light. 

•  Nacreous:  Translucent,  greyish-white,  with  pearly 
lustre. 

Sebaceous:  Translucent,  yellowish  or  greyish- white. 

Butyrous:  Translucent  and  yellow. 

Ceraceous:  Translucent  and  wax-coloured. 

Opaque. 

Cretaceous:  Opaque  and  white,  chalky. 

Dull:  Without  lustre. 

Glistening:  Shining. 

Fluorescent. 

Iridescent. 

2.  Chromogenicity : 

Colour  of  pigment. 

Pigment  restricted  to  colonies. 

Pigment  restricted  to  medium  surrounding  colonies. 

Pigment  present  in  colonies  and  in  medium. 

Streak  or  Smear  Cultures. — 

Gelatine  and  A  gar. — Note  general  points  as  indi- 
cated under  plate  cultivations. 

Inspissated  Blood-serum. — Note  the  presence  or 
absence  of  liquefaction  of  the  medium.  (The  presence 
of  condensation  water  at  the  bottom  of  the  tube  must 
not  be  confounded  with  liquefaction  of  the  medium.) 

All  Oblique  Tube  Cultures.— 

1.  Colonies  Discrete:  Size,  shape,  etc.,  as  for  plate 
cultivations  (vide  page  208). 

2.  Colonies  Confluent:  Surface  elevation  and  char- 
acter of  edge,  as  for  plate  cultivations  (vide  page  209). 

Chromogenicity:    As  for  plate  cultures   (supra). 


GELATINE    STAB    CULTURES.  215 

Gelatine  Stab  Cultures.— 

(A)  Surface  Growth. — As  for  individual  colonies  in 
plate  cultures  (vide  page  209). 

(B)  Line  of  Puncture. — 

Filiform:   Uniform   growth,    without   special   char- 
acters (Fig.  123,  a). 


Fig.  123. — Stab  cultivations — types  of  growth  :   a,  Filiform;  &,  beaded;   c, 
echinate  ;  d,  villous  ;  <?,  arborescent. 


Nodose:  Consisting  of  closely  aggregated  colonies. 
Beaded:  Consisting  of  loosely  placed  or  disjointed 
colonies  (Fig.  123,  b). 

Papillate:  Beset  with  papillate  extensions. 
Echinate :  Beset  with  acicular  extensions  (Fig.  1 23,  c) . 


21 6  METHODS   OF   IDENTIFICATION. 

Villous:  Beset  with  short,  undivided,  hair-like 
extensions  (Fig.  123,  d).  . 

Plumose:  A  delicate  feathery  growth. 

Arborescent:  Branched  or  tree-like,  beset  with 
branched  hair-like  extensions  (Fig.  123,  e). 


Fig.  124. — Stab  cultivations — types  of  growth:  /,   Crateriform  ;   g,  saccate; 
k,  infundibuliform  ;  j,  napiform  ;  k,  fusiform  ;  /,  stratiform. 

(C)  Area  of  Liquefaction  (if  present). — 

Crateriform:  A  saucer-shaped  liquefaction  of  the 
gelatine  (Fig.  124,  /). 

Saccate:  Shape  of  an  elongated  sack,  tubular, 
cylindrical  (Fig.  124,  g). 

Infundibuliform:  Shape  of  a  funnel,  conical  (Fig. 
124,  h). 


UTMUS   MILK   CULTIVATIONS.  217 

Napiform:  Shape  of  a  turnip  (Fig.  124,  ;). 

Fusiform:  Outline  of  a  parsnip,  narrow  at  either 
end,  broadest  below  the  surface  (Fig.  124,  k). 

Stratiform:  Liquefaction  extending  to  the  walls  of 
the  tube  and  downwards  horizontally  (Fig.  1 24,  /) . 

(D)  Character  of  the  Liquefied  Gelatine. — 

1.  Pellicle  on  surface. 

2.  Uniformly  turbid. 

3.  Granular. 

4.  Mainly  clear,  but  containing  flocculi. 

5.  Deposit  at  apex  of  liquefied  portion. 

(E)  Production  of  Gas  Bubbles. 
Shake  Cultures.— 

1.  Presence  or  absence  of  liquefaction. 

2.  Production  of  gas  bubbles. 

3.  Bulk  of  growth  at  the  surface — aerobic. 

4.  Bulk  of  growth  in  depths — anaerobic. 

Fluid  Media. 

1.  Surface  of  the  Liquid. — 

Presence  or  absence  of  froth  due  to  gas  bubbles. 
Presence  or  absence  of  pellicle  formation. 
Character  of  pellicle. 

2.  Body  of  the  Liquid.— 
Uniformly  turbid. 
Flocculi  in  suspension. 
Granules  in  suspension. 

Clear,  with  precipitate  at  bottom  of  tube. 
Colouration  of  fluid,  presence  or  absence  of. 

3.  Precipitate. — 
Character. 
Amount. 
Colour. 

Litmus  Milk  Cultivations. — 

{Unaltered. 
Acid. 
Alkaline. 


2l8  METHODS   OF    IDENTIFICATION. 

2.  Odour. 

3.  Formation  of  gas. 

c  Unaltered. 

4.  Consistency:  <  Digested  (?  character  of  solution) 

I  Coagulated. 

rA1        ^,  ( hard:  solid. 

5.  Clot:  Character^  ... 

\  soft:  flocculent. 

(a)  Coagulum  undissolved. 
(6)  Coagulum  finally  digested,  completely:  in- 
completely. 

Resulting  solution,  clear:  turbid. 
f  Abundant. 

nru  Scanty. 

6.  Whey:  1  J 

Clear. 

I  Turbid. 


BY  MICROSCOPICAL  METHODS. 

Preparations  must  be  made  from  the  cultivations 
at  intervals  of,  say,  twenty-four  hours,  during  the 
period  they  are  under  observation,  and  examined — 

(A)  Living. — 1.  In  hanging  drop,  to  determine  mo- 
tility  or  non-motility. 

In  this  connection  it  must  be  remembered  that 
under  certain  conditions  as  to  environment  (e.  g.,  cold, 
heat,  light,  unsuitable  medium,  etc.)  motile  bacilli 
may  fail  to  exhibit  activity.  No  organism,  therefore, 
should  be  recorded  as  non-motile  from  one  observation 
only;  a  series  of  observations  at  different  ages  and 
under  varying  conditions  should  form  the  basis  of  an 
opinion  as  to  the  absence  of  true  locomotion. 

Size. — In  the  case  of  non-motile  or  sluggishly  motile 
organisms,  endeavour  to  measure  several  individuals  in 
each  hanging  drop  by  means  of  the  eyepiece  microm- 
eter, and  average  the  results. 

If  the  organism  is  one  which  forms  spores,  ob- 
serve— 


BY   MICROSCOPICAL   METHODS.  2 19 

(a)  Spore  Formation. — Prepare  hanging-drop  culti- 
vations (vide  page  69)    from  vegetative  forms  of  the 
organism,  adding  a  trace  of  magenta  solution  (0.5  per 
cent.)  to  the  drop,  on  the  point  of  the  platinum  needle, 
to  facilitate  the  observation  of  the  phenomenon  by 
rendering  the  bacilli  more  distinct. 

Place  the  preparation  on  the  stage  of  the  micro- 
scope; if  necessary,  using  a  warm  stage. 

Arrange  illumination,  etc.,  and  select  a  solitary 
bacillus  for  observation,  by  the  help  of  the  ^-inch 
lens. 

Substitute  the  ^-inch  oil-immersion  lens  for  the 
sixth,  and  observe  the  formation  of  the  spore;  if 
possible,  measure  any  alteration  in  size  which  may 
occur  by  means  of  the  Ramsden  micrometer. 

(b)  Spore  Germination. — In  a  similar  manner  prepare 
hanging-drop    cultivations    from    old    cultivations    in 
which  no  living  vegetative  forms   are  present,   and 
observe  the  process  of  germination. 

The  comfort  of  the  microscopist  is  largely  enhanced 
in  those  cases  where  the  period  of  observation  is  at  all 
lengthy,  by  the  use  of  some  form  of  eye  screen  before 
the  unemployed  eye,  such  as  is  figured  on  page  59 
(Fig.  41). 

If  it  is  impossible  to  carry  out  the  method  suggested 
above,  proceed  as  follows: 

(a)  Spore  Formation. — Plant  the  organism  in  broth 
and  incubate  under  optimum  conditions. 

At  regular  intervals,  say  every  thirty  minutes,  re- 
move a  loopful  of  the  cultivation  and  prepare  a  cover- 
slip  film  preparation. 

Fix,  while  still  wet,  in  the  corrosive  sublimate  fixing 
solution. 

Stain  with  aniline  gentian  violet,  and  partially  de- 
colourise with  2  per  cent,  acetic  acid. 

Mount  and  number  consecutively. 

(b)  Spore  Germination. — Expose  a  thick  emulsion  of 


220  METHODS   OF   IDENTIFICATION. 

the  spores  to  a  temperature  of  80°  C.  for  ten  minutes 
in  the  differential  steriliser  (vide  page  202). 

Transfer  the  emulsion  to  a  tube  of  sterile  nutrient 
broth  and  incubate. 

Remove  specimens  from  the  tube  culture  at  intervals 
of,  say,  five  minutes. 

Fix,  stain,  etc.,  as  under  (a),  and  examine. 

(B)  Fixed. — 2.  In  stained  preparations. 

(a)  To  determine  points  in  morphology: 

Shape  (vide  classification,  page  in). 

Size: 

(a)  Prepare    cover-slip    film    preparations    at    the 
various  ages,  and  fix  by  exposure  to  a  temperature 
of  115°  C.  for  twenty  minutes  (vide  page  75). 

(b)  Stain  the  preparations  by  Gram's  method  (if 
applicable)  or  with  dilute  carbol-fuchsin,  and  mount 
in  the  usual  way. 

(c)  Measure  (vide  page  63)  some   twenty-five  indi- 
viduals in  each  film  by  means  of  the  Ramsden's  or 
the  stage  micrometer  and  average  the  result. 

Pleomorphism :  If  noted,  record — 
The  predominant  character  of  the  variant  forms. 
On  what  medium  or  media  they  are  observed. 
(b)  To  demonstrate  details  of  structure: 
Flagella:  If  noted,  record- 
Method  of  staining  (vide  page  87). 
Position  and  arrangement  (vide  page  115). 
Number. 

Spores:  If  noted,  record — 
Method  of  staining. 
Shape. 
Size. 

Position  within  the  parent  cell. 
Condition,   as   to  shape,    of   the   parent   cell    (vide 
page  74). 

On  what  medium  they  are  best  observed. 
Age  of  medium. 


BY    CHEMICAL   METHODS.  221 

Conditions  of  environment  as  to  temperature,  atmos- 
phere. 

Method  of  germination  (vide  page  119). 

Involution  Forms:  If  noted,  record — 

Method  of  staining. 

Character  (e.  g.,  if  living  or  dead). 

Shape. 

On  what  medium  they  are  observed. 

Age  of  medium. 

Environment. 

Metachromatic  Granules:  If  noted,  record — 

Method  of  staining. 

Character  of  granules. 

Number  of  granules. 

Colour  of  granules. 

3.  Staining  Reactions. — 

1.  Gram's  Method. — Decolourised  or  retain  the  stain. 

2.  Neisser's  Method. — If  granules  are  noted,  record — 

1.  Position. 

2.  Number. 

3.  Ziehl-Neelsen's    Method. — Decolourised    or    acid- 
fast. 

4.  Simple  Aniline  Dyes. — (Noting  those  giving  the 
best  results,  with  times  of  staining.) 

Methylene-blue 

Fuchsm  aiid  the.r  modifications> 

Gentian  violet 

Thionine  blue 


BY  CHEMICAL  METHODS. 

Test  cultivations  of  the  organism  for  the  presence  of — 

Soluble  enzymes — proteolytic,  diastatic,  invertin. 

Organic  acids — (a)  quantitatively — i.  e.,  estimate 
the  total  acid  production;  (6)  qualitatively  for 
formic,  acetic,  propionic,  butyric,  lactic. 

Ammonia. 


222  METHODS    OF    IDENTIFICATION. 

Alcohol — ethyl  alcohol,  aldehyde,  acetone. 

Aromatic  products — indol,  phenol. 

Soluble  pigments. 

Test  the  power  of  reducing  (a)  colouring  matters, 
(6)  nitrates  to  nitrites. 

Investigate  the  gas  production — H2S,  CO2,  H.  Esti- 
mate the  ratio  between  the  last  two  gases. 

Prepare  all  cultivations  for  these  methods  of  ex- 
amination under  optimum  conditions,  previously  deter- 
mined for  each  of  the  organisms  it  is  intended  to 
investigate,  as  to 

(a)  Reaction  of  medium; 
(6)  Incubation  temperature; 
(c)  Atmospheric  environment. 

and  keep  careful  records  of  these  points,  and  also  of 
the  age  of  the  cultivation  used  in  the  final  examina- 
tion. 

Examine  the  cultivations  for  the  various  products 
of  bacterial  metabolism  after  forty-eight  hours'  growth, 
and  never  omit  to  examine  "  control  "  (uninoculated) 
tube  or  flask  of  medium  kept  for  a  similar  period 
under  identical  conditions.  If  the  results  are  nega- 
tive, test  further  cultivations  at  three  days,  five  days, 
and  ten  days. 

1.  Enzyme  Production. — 

(A)  Proteolytic  Enzymes. — (Convert  proteids  into 
peptones  and  propeptones;  e.  g.,  B.  pyocyaneus.) 

Media  Required: 

Blood-serum  and  milk-serum  which  have  been  carefully 

filtered  through  a  porcelain  candle. 
Reagents  Required: 

Ammonium  sulphate. 

^  caustic  soda  solution. 

Copper  sulphate,  i  per  cent,  aqueous  solution. 

METHOD. — 

i.  Prepare  cultivations  in  bulk  (50  c.c.)  in  a  flask 
and  incubate. 


BY   CHEMICAL   METHODS.  223 

2.  Add  60  grammes  of  ammonium  sulphate  to  40 
c.c.  of  the  cultivation,  and  warm  to  50°  C.  for  half 
an  hour.     (This  precipitates  the  proteid  bodies.) 

3.  Filter. 

Test  the  filtrate  for  propeptones  and  peptones. 
Make  the  filtrate  strongly  alkaline  with  caustic  soda. 
Add  a  few  drops  of  copper  sulphate  solution. 
Violet  colour  =  peptones. 

(B)  Diastatic  Enzymes. — (Convert  starch  into  sugar; 
e.  g.,  B.  subtilis.) 

Medium  Required: 

Inosite-free  bouillon. 
Reagents  Required: 

Starch. 

Thymol. 

Fehling's  solution. 

METHOD.— 

1.  Prepare  tube  cultivation  and  incubate. 

2.  Prepare  a  thin  starch  paste  and  add  2  per  cent, 
thymol  to  it. 

3.  Mix  equal  parts  of  the  cultivation  to  be  tested 
and  the  starch  paste,  and  place  in  the  incubator  at 
37°  C.  for  six  to  eight  hours. 

4.  Filter. 

Test  the  filtrate  for  sugar. 

Boil  some  of  the  Fehling's  solution  in  a  test-tube. 

Add  the  filtrate  drop  by  drop  until,  if  necessary,  a 
quantity  has  been  added  equal  in  amount  to  the 
Fehling's  solution  employed,  keeping  the  mixture  at 
the  boiling-point  during  the  process. 

Yellow  or  orange  precipitate  =  sugar. 

(C)  Invertin    Enzymes. — (Convert    saccharose    into 
glucose;  e.  g.,  B.  fluorescens  liquefaciens.) 

Medium  Required: 

Inosite-free  bouillon. 
Reagents  Required: 

Cane  sugar,  2  per  cent,  aqueous  solution. 

Carbolic  acid. 


224  METHODS  OF  IDENTIFICATION. 

METHOD. — 

1.  Prepare  tube  cultivations  and  incubate. 

2.  Add  2  per  cent,  of  carbolic  acid  to  the  sugar 
solution. 

3.  Mix  equal  quantities  of  the  carbolised  sugar  solu- 
tion  and   the   cultivation  in   a   test-tube;   allow  the 
mixture  to  stand  for  several  hours. 

4.  Filter. 

Test  the  nitrate  for  glucose  as  in  the  preceding 
section. 

(D)  Rennet  and  "Lab  "  Enzymes. — (Coagulate  milk 
independently  of  the  action  of  acids;  e.  g.,  B.  pro- 
digiosus.) 

Media  Required: 

Inosite-free  bouillon. 
Litmus  milk. 

METHOD. — 

1.  Prepare  tube  cultivations  and  incubate. 

2.  After  incubation  heat  the  cultivation  to  55°  C. 
for  half  an  hour,  to  sterilise. 

3.  By  means  of  a  sterile  pipette  run  5  c.c.  of  the 
cultivation  into  each  of  three  tubes  of  litmus  milk. 

4.  Place  in  the  cold  incubator  at  22°  C.  and  examine 
each  day  for  ten  days. 

Absence  of  coagulation  at  the  end  of  that  period 
will  indicate  absence  of  rennet  ferment  formation. 
2.  Acid  Production. — 
(a)  Quantitative. — 

Medium  Required: 

Sugar  (glucose)  bouillon  (vide  page  142)  of  known  "  opti- 
mum "  reaction. 
Apparatus  and  Reagents  Required: 

As  for  estimating  reaction  of  media  (vide  page  129). 

METHOD.— 

1.  Prepare  cultivation  in  bulk  (100  c.c.)  in  a  flask. 

2.  After  suitable  incubation,  heat  in  the  steamer  at 
1 00°  C.  for  thirty  minutes  to  sterilise. 


BY    CHEMICAL    METHODS.  225 

3.  Determine  the  litre  of  the  medium  as  described 
in  the  preparation  of  nutrient  media  (vide  page  130). 

4.  The  difference  between  the  original  titre  of  the 
medium  and  that  now  estimated  gives  the  total  acid 
production  in  terms  of  normal  NaOH. 

NOTE. — If  the  growth  is  very  heavy  it  may  be  a 
difficult  matter  to  determine  the  end-point.  The 
cultivation  should  then  be  filtered  through  a  Berkfeld 
filter  candle  previous  to  step  2,  and  the  filtrate  em- 
ployed in  the  titration. 

(b)  Qualitative  (of  all  the  organic  acids  present).— 

Medium  Required: 

Sugar  (glucose  or  lactose)  bouillon  as  in  quantitative  ex- 
amination. 
Reagents  Required: 

Hydrochloric  acid,  concentrated. 

Sulphuric  acid,  concentrated  (pure). 

Sulphuric  acid,  25  per  cent,  solution. 

Ammonia. 

Ammonium  sulphide. 

Baryta  water. 

Sodium  carbonate,  saturated  aqueous  solution. 

Absolute  alcohol. 

Ether. 

Calcium  chloride. 

Zinc  oxide. 

Permanganate  of  potash,  4  per  cent,  aqueous  solution. 

Schiff's  reagent. 

Arsenious  oxide. 

Ferric  chloride,  4  per  cent,  aqueous  solution. 

Cobalt  nitrate,  2  per  cent,  aqueous  solution. 

Silver  nitrate,  i  per  cent,  aqueous  solution. 

Lugol's  iodine  (vide  page  94). 

Cane  sugar,  10  per  cent,  aqueous  solution. 

Hard  paraffin  wax  (melting-point  about  52°  C.). 

METHOD. — 

1.  Prepare  cultivation  in  bulk  (500  c.c.)  in  a  litre 
flask  and  add  sterilised  precipitated  chalk,  10  grammes. 
Incubate  at  the  optimum  temperature. 

2.  After  incubation  throw  a  piece  of  paraffin  wax 
(about  a  centimetre  cube)  into  the  cultivation  and 
connect  up  the  flask  with  a  condenser. 

15 


226 


METHODS    OF   IDENTIFICATION. 


The  paraffin,  which  liquefies  and  forms  a  thin 
layer  on  the  surface  of  the  fluid,  is  necessary  to  pre- 
vent the  cultivation  frothing  up  and  running  un- 
altered through  the  condenser  during  the  subsequent 
process  of  distillation. 

3.  Distil  over  200  to  300  c.c. 

Use  a  rose-top  burner  to  minimise  the  danger  of 


Fig.  125. — Arrangement  of  distillation  apparatus  for  acids,  etc. 


cracking  the  flask;  and  to  the  same  end,  well  agitate 
the  contents  of  the  flask  to  prevent  the  chalk  settling. 

The  distillate  "A"  will  contain  alcohol,  etc.  (vide 
page  229);  the  residue  "a"  will  contain  the  volatile 
and  fixed  acids. 

4.  Disconnect  the  flask  and  filter  the  chalk  from  the 


contained    residue;    add    10    c.c. 
acid  to  the  filtrate;  mix  well. 


cone,    hydrochloric 


BY    CHEMICAL    METHODS. 


227 


5.  Precipitate  the  calcium  by  adding  sodium  car- 
bonate solution,  until  alkaline. 

6.  Boil  thoroughly  (to  ensure  complete  precipitation 
of  lime)  and  filter. 

7.  Add  20  c.c.  sulphuric  acid  (25  per  cent.)  to  the 
filtrate   (this  liberates  the  volatile  acids),   and  distil 
as  completely  as  possible. 

DISTILLATE  "B." 

(Volatile  Acids.) 


1.  Saturate  with  baryta  water  to  alkalinity, 

and  evaporate  to  dryness. 

2.  Add  20  c.c.  absolute  alcohol  and  allow 

to  stand,  with  frequent  stirring,  for 
two  to  three  hours. 

3.  Filter  and  wash  with  alcohol. 


FILTRATE 

may  contain  barium  propionate, 
barium  butyrate. 


1.  Evaporate  to  dryness. 

2.  Dissolve  residue  in  150  c.c.  water. 

3.  Saturate  with  calcium  chloride. 

4.  Distil. 

5.  Test  distillate  for  butyric  acid  : 

Add  3  c.  c.  alcohol  and  4  drops  con- 
centrated sulphuric  acid. 
Smell  of  pineapple  =  butyric 

acid. 

Propionic  acid  in  small  quantities  can- 
not be  distinguished  from  butyric 
acid  by  tests  within  the  scope  of 
the  bacteriological  laboratory. 


RESIDUE 

may  contain  barium  acetate, 
barium  formate. 


1.  Evaporate  off   a  cohol    and   dissolve 

up  the  residue  on  the  filter  in  hot 
water. 

2.  Divide  the  solution  into  four  portions : 

(a)  Add  ferric  chloride  solution. 

Brown  colour  =  acetic  or  for- 
mic acids. 

(b)  Add  silver  nitrate  solution. 

White  flocculent  precipitate 
(soluble  in  hot  water  and  sep- 
arating in  spangles  when 
cool)  =  acetic  acid. 
Add  silver  nitrate  solu^n  ;  then 
add  one  drop  ammonia  water, 
and  boil. 

Black  precipitate  of    metallic 
silver  =  formic  acid. 

(c)  Evaporate  to  dryness  ;  mix  with 

equal  quantity  of  arsenious 
oxide  and  heat  on  platinum 
foil. 

Unpleasant  smell  of  cacodyl 
=  acetic  acid. 

(d)  Add  a    few  drops  of    mercuric 

chloride  solution  in  test-tube, 
and  heat  to  70°  C. 
Precipitate  of  mercurous  chlo- 
ride and  the  formation,  after 
a  long  time,  of  a  metallic 
mirror  =  formic  acid. 


The  distillate  "B"  may  contain  acetic,  propionic, 
formic,  or  butyric  acid. 


228 


METHODS    OF   IDENTIFICATION. 


The  residue  "b"  may  contain  lactic,  oxalic,  succinic, 
glycocholic,  taurocholic,  cholic,  benzoic,  hippuric, 
tannic,  or  gallic  acid. 


RESIDUE  «b.' 
(Fixed  Acids.) 


1.  Evaporate  the  remainder  of  the  residue 

to  a  syrup. 

2.  Extract  with    ether   by  agitation   in  a 

separatory  funnel.      (This  dissolves 
out  the  fixed  organic  acids.) 

3.  'Evaporate  ethereal  extract  to  a  syrup. 

(No    residue  =   absence    of    lactic, 
oxalic,  succinic  acids.) 

4.  Add  100  c.c.  water  and  mix  thoroughly. 

5.  Add  excess  of  zinc  oxide  and  heat  nearly 

to  boiling.     Filter. 


FILTRATE 


(a)  Test  for  cholic  acid  series  : 

1.  To  6  c.c.  filtrate  add  4  c.c.  cone. 

sulphuric  acid  ;  then  add  i 
drop  cane  sugar  solution 
and  warm  to  75°  C. 
Crimson  colour  =  glyco- 
cholic,  taurocholic,  or 
cholic  acids. 

2.  To  a  portion  of  the  filtrate  add 

Lugol's  iodine. 
Blue  colour  (resembling 
that   of    the    starch  -iodo 
compound)  =  cholic  acid. 

(b)  Test  for  lactic  acid  : 

1.  Acidify  with  hydrochloric  acid. 

2.  Add  ammonia  water  in  slight  ex- 

cess and  boil  off  the  excess. 

3.  Add  cobalt  nitrate  solution. 

Violet  colour  (or  if  in  suffi- 
cient   quantity  crystalline 
precipitate)  =  lactic  acid. 
Or— 

1.  Evaporate  bulk  of  filtrate  to  dry- 

ness. 

2.  Dissolve  in  10  c.c.  hot  water. 

3.  Allow  to  crystallise — with  con- 

centration if  necessary. 
Crystals  of  zinc  lactate  = 
lactic  acid. 

( Do  not  confuse  with  zinc 
sulphate,  which  will 
probably  be  present 
also.) 


RESIDUE. 


Dissolve  in  hydrochloric  acid  from  off 
the  filter. 

(a)  Test  for  oxalic  acid  : 

1.  Neutralise    with    ammonia    till 

faintly  alkaline. 

2.  Add  calcium  chloride  solution. 

White  precipitate  =  cal- 
cium oxalate.  No  precipi- 
tate =  absence  of  oxalic 
acid. 

(b)  Test  for  succinic,  benzoic,  or  hip- 

puric, salicylic,  tannic,  or  gallic 
acid  : 

1.  Neutralise  with    ammonia   and 

boil  off  the  excess. 

2.  Add  ferric  chloride  solution  on  a 

glass  rod. 

Red»brown  colouration  or 
precipitate  =  succinic  acid. 

Buff  colouration  or  precipi- 
tate =  benzoic  or  hippuric 
acid. 

Violet  colouration  or  pre- 
cipitate =  salicylic  acid. 

Inky-black  colouration  or 
precipitate  =  tannic  or 
gallic  acid. 


8.  Use  50  c.c.  of  the  distillate  "B"  for  titrations. 
This  will  give  the  amount  of  volatile  acid  formation. 


BY    CHEMICAI,    METHODS.  229 

3.  Ammonia  Production. — 

Medium  Required: 

Nutrient  bouillon. 
Reagent  Required: 

Nessler  reagent. 

METHOD.— 

1.  Prepare  cultivation  in  bulk   (100  c.c.)  in  a  250 
c.c.  flask  and  incubate  together  with  a  control  flask. 

Test  the  cultivation  and  the  control  for  ammonia 
in  the  following  manner: 

2.  To  each  flask  add  2  grammes  of  calcined  mag- 
nesia, then  connect  up  with  condensers  and  distil. 

3.  Collect  50  c.c.  distillate,  from  each,  in  a  Nessler 
glass. 

4.  Add  to  each  i  c.c.  Nessler  reagent  by  means  of  a 
clean  pipette. 

A  yellow  colour  =  ammonia. 

The  depth  of  colour  is  proportionate  to  the  amount 
present. 

4.  Alcohol,  etc.,  Production. — Divide  the  distillate 
"A"  obtained  in  the  course  of  a  previous  experiment 
(vide  page  226,  step  3)  into  four  portions  and  test  for 
the  production  of  alcohol,  acetaldehyde,  acetone. 

1.  Add  Lugol's  iodine,  then  a  little  NaOH  solution, 
and  stir  with  a  glass  rod. 

Pale  yellow  crystalline. precipitate  of  iodoform,  with 
its  characteristic  smell,  indicates  alcohol,  aldehyde, 
or  acetone. 

The  precipitate  may  be  absent  even  when  the  odour 
is  pronounced. 

2.  Add  Schiff's  reagent. 

Violet  or  red  colour  =  aldehyde. 
.3.  To  10  c.c.  of  solution  add  5  c.c.  cone,  sulphuric 
acid,  and  i  c.c.  of  potassium  permanganate  solution. 
After  an  interval  of  five  minutes  add  Schiff's  reagent. 
Red  colour  (due  to  oxidation  of  magenta  in  reagent) 
=  aldehyde  from  alcohol. 


230  METHODS    OP   IDENTIFICATION. 

4.  Make  the  solution  strongly  alkaline  with  ammonia. 
Add   gradually   a   solution   of   iodine   in    ammonium 
iodide.     A  black  precipitate  of  nitrogen  iodide  forms, 
which  quickly  disappears  on  shaking.     As  soon  as  the 
precipitate  tends  to  become  permanent,  it  will  change 
to  iodoform  if  acetone  is  present. 

5.  Indol  Production. — 

Media  Required: 

Inosite-free  bouillon  (vide  page  141). 

Or  peptone  water  (vide  page  168). 
Reagents  Required: 

Sulphuric  acid,  concentrated  pure. 

Sodium  nitrite,  o.oi  per  cent,  aqueous  solution. 

METHOD. — 

1.  Prepare    several    test-tube    cultivations    of    the 
organism  to  be  tested,  and  incubate. 

Test  for  indol  by  means  of  the  nitroso-indol  reaction, 
in  the  following  manner.  (If  the  culture  has  been 
incubated  at  37°  C.,  it  must  be  allowed  to  cool  to  the 
room  temperature  before  applying  the  test.) 

2.  Remove  the  cotton- wool  plug  from  the  tube,  and 
run  in  i  c.c.  sulphuric  acid  by  means  of  a  sterile  pipette. 
Place  the  tube  upright  in  a  rack,  and  allow  it  to  stand, 
if  necessary,  for  ten  minutes. 

A  rose-pink  or  red  colour  =  indol  (plus  a  nitrite). 

3.  If  the  colour  of  the  medium  remains  unaltered, 
add  2  c.c.  sodium  nitrite  solution,  and  again  allow 
the  culture  to  stand  for  ten  minutes. 

Red  colouration  —  indol. 

NOTE. — In  place  of  performing  the  test  in  two 
stages  as  given  above,  2  c.c.  concentrated  commercial 
sulphuric,  hydrochloric,  or  nitric  acid,  all  of  which 
hold  a  trace  of  nitrite  in  solution,  may  be  run  into 
the  cultivation.  The  development  of  a  red  colour 
within  twenty  minutes  will  indicate  the  presence  of 
indol. 


BY  CHEMICAL   METHODS.  231 

5a.  Phenol  Production. — 

Medium  Required: 

Nutrient  bouillon. 
Reagents  Required: 

Hydrochloric  acid,  concentrated. 

Millon's  reagent. 

Ferric  chloride,  i  per  cent,  aqueous  solution. 

METHOD. — 

1.  Prepare  cultivation  in  a  Bohemian  flask  contain- 
ing at  least  50  c.c.  of  medium,  and  incubate. 

Test  for  phenol  in  the  following  manner : 

2.  Add  5  c.c.  hydrochloric  acid  to  the  cultivation 
and  connect  up  the  flask  with  a  condenser. 

3.  Distil  over   15  to  20  c.c.     Divide  the  distillate 
into  two  portions. 

4.  Test  one  portion  by  adding  0.5  c.c.  Millon's  re- 
agent and  boiling. 

A  red  colour  —  phenol. 

5.  Test  the  other  portion  by  adding  about  0.5  c.c. 
ferric  chloride  solution. 

A  violet  colour  =  phenol. 

NOTE. — If  both  indol  and  phenol  appear  to  be  present 
in  cultivations  of  the  same  organism,  it  is  well  to 
separate  them  before  testing.  This  may  be  done  in 
the  following  manner: 

1.  Prepare  inosite-free  bouillon  cultivation,  say  200 
or  300  c.c.,  in  a  flask  as  before. 

2.  Add  50  to  60  c.c.  hydrochloric  acid  and  connect 
up  the  flask  with  a  condenser. 

3.  Distil  over  50  to  70  c.c. 

Distillate  will  contain  both  indol  and  phenol. 

4.  Render  the  distillate  strongly  alkaline  with  caustic 
potash  and  redistil. 

Distillate  will  contain  indol ;  residue  will  contain 
phenol. 

5.  Test  the  distillate  for  indol  (vide  ante). 


232  METHODS    OF    IDENTIFICATION. 

6.  Saturate   the  residue,    when   cold,    with   carbon 
dioxide  and  redistil. 

7.  Test  this  distillate  for  phenol  (vide  ante). 

6.  Pigment  Production. — 

1.  Prepare  tube  cultivations  upon  the  various  media 
and  incubate   under  varying  conditions   as   to   tem- 
perature (at  37°  C.  and  at  20°  C.),  atmosphere  (aerobic 
and  anaerobic),  and  light  (exposure  to  and  protection 
from). 

Note  the  conditions  most  favorable  to  pigment 
formation. 

2.  Note  the  solubility  of  the  pigment  in  various 
solvents,  such  as  water,  hot  and  cold,  alcohol,  ether, 
chloroform,  benzol,  carbon  bisulphide. 

3.  Note  the  effect  of  acids  and  alkalies  respectively 
upon  the  pigmented  cultivation,  or  upon  solutions  of 
the  pigment. 

4.  Note  spectroscopic  reactions. 

7.  Reducing  Agent  Formation. — 

(a)  Colour  Destruction. — 

1.  Prepare   tube   cultivations   in   nutrient   bouillon 
tinted    with    litmus,    rosolic    acid,    neutral    red,    and 
incubate. 

2.  Examine  the  cultures  each  day  and  note  whether 
any   colour   change   occurs. 

(b)  Nitrates  to  Nitrites.— 

Medium  Required: 

Nitrate  bouillon  (vide  page  143). 

Or  nitrate  water  (vide  page  169). 
Reagents  Required: 

Sulphuric  acid  (25  per  cent.). 

Metaphenylene  diamine,  5  per  cent,  aqueous  solution. 

METHOD.— 

i.  Prepare  tube  cultivations  and  incubate  together 
with  control  tubes  (i.  e.t  uninoculated  tubes  of  the 
same  medium,  placed  under  identical  conditions  as  to 
environment). 


BY   CHEMICAL    METHODS.  233 

This  precaution  is  necessary  as  the  medium  is  liable 
to  take  up  nitrites  from  the  atmosphere,  and  an 
opinion  as  to  the  absence,  of  nitrites  in  the  cultiva- 
tion is  often  based  upon  an  equal  colouration  of  the 
medium  in  the  control  tube. 

Test  both  the  culture  tube  and  the  control  tube 
for  the  presence  of  nitrites. 

2.  Add  a  few  drops  of  sulphuric  acid  to  the  medium 
in  each  of  the  tubes. 

3.  Then  run  in  2  or  3  c.c.  metaphenylene  diamine 
into  each  tube. 

A  brownish-red  colour  =  nitrites. 
The  depth  of  colour  is  proportionate  to  the  amount 
present. 

8.  Gas  Production. — 

(A)  Carbon  Dioxide  and ( Hydrogen. — 

Apparatus  Required: 

Fermentation   tubes    (vide    page    24)    containing    sugar 
bouillon  (glucose,  lactose,  etc.)-    The  medium  should  be 
prepared  from  inosite-free  bouillon  (vide  page  141). 
Reagent  Required: 
^  caustic  soda. 

METHOD.— 

1.  Inoculate  the  surface  of  the  medium  in  the  bulb 
of  a  fermentation  tube  and  incubate. 

2.  Mark  the  level  of  the  fluid  in  the  closed  branch 
of  the  fermentation  tube,  at  intervals  of  twenty-four 
hours,    and   when   the   evolution   of  gas   has   ceased, 
measure  the  length  of  the  column  of  gas  with  the 
millimetre  scale. 

Express  this  column  of  gas  as  a  percentage  of  the 
entire  length  of  the  closed  branch. 

3.  To  analyse    the  gas  and  to  determine  roughly 
the    relative  proportions  of    CO2  and  H,  proceed  as 
follows : 

Fill  the  bulb  of  the  fermentation  tube  with  caustic 
soda  solution. 


234  METHODS  OF  IDENTIFICATION. 

Close  the  mouth  of  the  bulb  with  a  rubber  stopper. 

Alternately  invert  and  revert  the  tube  six  or  eight 
times,  to  bring  the  soda  solution  into  intimate  contact 
with  the  gas. 

Return  the  residual  gas  to  the  end  of  the  closed 
branch,  and  measure. 

The  loss  in  volume  of  gas  =  carbon  dioxide. 

The  residual  gas  =  hydrogen. 

Transfer  gas  to  the  bulb  of  the  tube,  and  explode  it 
by  applying  a  lighted  taper. 

(B)  Sulphuretted  Hydrogen.— 

Media  Required: 

Peptone  iron  solution  (vide  page  168). 
Peptone  lead  solution. 

1.  Inoculate  tubes  of  media,  and  incubate  together 
with  control  tubes. 

2.  Examine  from  day  to  day,  at  intervals  of  twenty- 
four  hours. 

The  liberation  of  the  H2S  will  cause  the  yellowish- 
white  precipitate  to  darken  to  a  brownish-black,  or 
jet  black,  the  depth  of  the  colour  being  proportionate  to 
the  amount  of  sulphuretted  hydrogen  present. 

Quantitative :  For  exact  quantitative  analyses  of  the 
gases  produced  by  bacteria  from  certain  media  of 
definite  composition,  the  methods  devised  by  Pakes 
must  be  employed,  as  follows: 

Apparatus  Required: 

Bohemian  flask  (300  to  1500  c.c.  capacity)  containing 
from  100  to  400  c.c.  of  the  medium.  The  mouth  of 
the  flask  is  fitted  with  a  perforated  rubber  stopper,  car- 
rying an  L -shaped  piece  of  glass  tubing  (the  short  arm 
passing  just  through  the  stopper).  To  the  long  arm  of 
the  tube  is  attached  a  piece  of  pressure  tubing  some 
8  cm.  in  length,  plugged  at  its  free  end  with  a  piece  of 
cotton- wool.  Measure  accurately  the  total  capacity  of 
the  flask  and  exit  tube,  also  the  amount  of  medium  con- 
tained. Note  the  difference. 

Gas  receiver.  This  is  a  bell  jar  of  stout  glass,  14  cm.  high 
and  9  cm.  in  diameter.  At  its  apex  a  glass  tube  is  fused 


BY  CHEMICAL  METHODS. 


235 


in.  This  rises  vertically  5  cm.,  and  is  then  bent  at  right 
angles,  the  horizontal  arm  being  10  cm.  in  length.  A 
three-way  tap  is  let  horizontally  into  the  vertical  tube 
just  above  its  junction  with  the  bell  jar. 

An  iron  cylinder  just  large  enough  to  contain  the  bell  jar. 

About  15  kilos  of  mercury. 

Melted  paraffin. 

An  Orsat-Lunge  working  with  mercury  instead  of  water, 
provided  with  two  gas  tubes  of  extra  length  (capacity 
1 20  and  60  c.c.  respectively  and  graduated  throughout, 
both  being  water-jacketed)  or  other  gas  analysis  appa- 
ratus, capable  of  deal- 
ing with  CO,  CO,,  O, 
H, and  N. 


METHOD.— 

1.  Inoculate    the    me- 
dium in  the  flask  in  the 
usual  manner,  by  means 
of    a     platinum    needle, 
taking  care  that  the  neck 
of  the  flask  and  the  rub- 
ber   stopper     are    thor- 
oughly   heated     in    the 
flame    before    and    after 
the  operation. 

2.  Fill  the  iron  cylinder  with  mercury. 

3.  Place    the    bell   jar    mouth    downwards    in  the 
mercury — first  seeing  that  there  is  free  communication 
between  the  interior  of  the  jar  and  the  external  air— 
and  suck  up  the  mercury  into  the  tap;  then  shut  off 
the  tap. 

4.  Plug  the  open  end  of  the  three-way  tap  with 
melted  wax. 

5.  Connect  up  the  horizontal  arm   of   the   culture 
flask  with  that  of  the  gas  receiver  by  means  of  the 
pressure  tubing  (after  removing  the  cotton-wool  plug 
from  the  rubber  tube),  as  shown  in  Fig,  127. 

6.  Give  the  three-way  tap  a  half  turn  to  open  com- 
munication between  flask  and  receiver,  and  seal  all 


Fig.  126. — Orsat-Lunge  gas  analysis 
apparatus. 


236 


METHODS    OF   IDENTIFICATION. 


joints  by  coating  with  a  film  of  melted  wax.  When 
the  tap  is  turned,  the  mercury  in  the  receiver  will 
naturally  fall. 

7.  Place    the    entire    apparatus    in    the   incubator. 
(Two  hours  later,  by  which  time  the  temperature  of 
the   apparatus   is   that   of   the   incubator,    mark   the 
height  of  the  mercury  on  the  receiver.) 

8.  Examine  the  apparatus  from  day  to  day  and  mark 
the  level  of  the  mercury  in  the  receiver  at  intervals 
of  twenty-four  hours. 

9.  When  the  evolution  of  gas  has  ceased,  remove 


Fig.  127. — Gas-collecting  apparatus. 

the  apparatus  from  the  incubator;  clear  out  the  wax 
from  the  nozzle  of  the  three-way  tap  (first  adjusting 
the  tap  so  that  no  escape  of  gas  shall  take  place)  and 
connect  it  with  the  Orsat. 

10.  Remove,  say,  100  c.c.  of  gas  from  the  receiver, 
reverse  the  tap  and  force  it  into  the  culture  flask. 
Remove  100  c.c.  of  mixed  gases  from  the  culture 
flask  and  replace  in  the  receiver. 

Repeat  these  processes  three  or  four  times  to  ensure 
thorough  admixture  of  the  contents  of  flask  and  re- 
ceiver. 


BY    CHEMICAI,   METHODS.  237 

1 1 .  Now  withdraw  a  sample  of  the  mixed  gases  into 
the  Orsat  and  analyse. 

In  calculating  the  results  be  careful  to  allow  for  the 
volume  of  air  contained  in  the  flask  at  the  commence- 
ment of  the  experiment. 

For  the  collection  of  gases  formed  under  anaerobic 
conditions  a  slightly  different  procedure  is  adopted: 

1.  Fix  a  culture  flask  (500  c.c.  capacity)  with  a  per- 
forated rubber  stopper  carrying  an  L-shaped  piece  of 
manometer  tubing,  each  arm  5  cm.  in  length. 

2.  Prepare  a  second  L-shaped  piece  of  tubing,  the 
short  arm  5  cm.  and  the  long  arm  20  cm.,  and  connect 
its  short  arm  to  the  horizontal  arm  of  the  tube  in  the 
culture  flask  by  means  of  a  length  of  pressure  tubing, 
provided  with  a  screw  clamp. 

3.  Fill   the    culture  flask    completely   with  boiling 
medium  and  pass  the  long  piece  of  tubing  through  the 
plug  of  an  Erlenmeyer  flask  (150  c.c.  capacity)  which 
contains  100  c.c.  of  the  same  medium. 

4.  Sterilise  these  coupled  flasks  by  the  discontinuous 
method,  in  the  usual  manner. 

Immediately  the  last  sterilisation  is  completed, 
screw  up  the  clamp  on  the  pressure  tubing  which  con- 
nects them,  and  allow  them  to  cool. 

As  the  fluid  cools  and  contracts  it  leaves  a  vacuum 
in  the  neck  of  the  flask  below  the  rubber  stopper. 

5.  To  inoculate  the  culture  flask,  withdraw  the  long 
arm  of  the  bent  tube  from  the  Erlenmeyer  flask  and 
pass  it  to  the  bottom  of  a  test-tube  containing  a  young 
cultivation   (in  a  fluid  medium  similar  to  that  con- 
tained in  the  culture  flask)  of  the  organism  it  is  desired 
to  investigate. 

6.  Slightly  release  the  clamp  on  the  pressure  tubing 
to  allow  4  or  5  c.c.  of  the  culture  to  enter  the  flask. 

7.  Clamp  the  rubber  tube  tightly;  remove  the  bent 
glass  tube  from  the  culture  tube  and  plunge  it  into  a 


238  METHODS    OF   IDENTIFICATION. 

flask  containing  recently  boiled  and  quickly  cooled  dis- 
tilled water. 

8.  Release  the  clamp  again  and  wash  in  the  remains 
of  the  cultivation  until  the  culture  flask  and  tubing 
are  completely  filled  with  water. 

9.  Clamp  the  rubber  tubing  tightly  and  take  away 
the  long-armed  glass  tubing. 

10.  Prepare   the   gas   receiver   as   in   the   previous 
method  (in  this  case,  however,  the  mercury  should  be 
warmed  slightly)  and  fill  the  horizontal  arm  of  the 
receiver  with  hot  water. 

1 1 .  Connect  up  the  culture  flask  with  the  horizontal 
arm  of  the  gas  receiver. 

12.  Remove  the  screw  clamp  from  the  rubber  tubing, 
adjust  the  three-way  tap,  seal  all  joints  with  melted 
wax,   and  incubate. 

13.  Complete  the  investigation  as  described  for  the 
previous  method. 

BY  PHYSICAL  METHODS, 

Examine  cultivations  of  the  organism  with  reference 
to  the  following  points: 
Atmosphere : 

(a)  In  the  presence  of  oxygen. 

(b)  In  the  absence  of  oxygen. 

(c)  In  the  presence  of  gases  other  than  oxygen. 
Temperature : 

(a)  Range. 

(b)  Optimum. 

(c)  Thermal  death-point: 

Moist:  Vegetative  forms. 

Spores. 
Dry:  Vegetative  forms. 

Spores. 

Reaction  of  medium. 
Resistance  to  lethal  agents: 
(a)  Desiccation. 


BY    PHYSICAL    METHODS.  239 

(b)  Light:  Diffuse. 

Direct. 
Primary  colours. 

(c)  Heat. 

(d)  Chemical  antiseptics  and  disinfectants. 
Vitality  in  artificial  cultures. 
Agglutination  reaction. 

I.  Atmosphere. — The  question  as  to  whether  the 
organism  under  observation  is  (a)  an  obligate  aerobe, 
(b)  a  facultative  anaerobe,  or  (c)  an  obligate  anaerobe 
is  roughly  decided  by  the  appearance  of  cultivations 
in  the  fermentation  tubes.  Obvious  growth  in  the 
closed  branch  as  well  as  in  the  bulb  will  indicate  that 
it  is  a  facultative  anaerobe;  whilst  growth  only  occur- 
ring in  the  bulb  or  in  the  closed  branch  shows  that  it 
is  an  obligate  aerobe  or  anaerobe  respectively.  This 
method,  however,  is  not  sufficiently  accurate  for  the 
present  purpose,  and  the  examination  of  an  organism 
with  respect  to  its  behaviour  in  the  absence  of  oxygen 
is  carried  out  as  follows: 

Apparatus  Required: 

Buchner's  tubes. 

Bulloch's  apparatus. 

Exhaust  pump. 

Pyrogallic  acid. 

Dekanormal  caustic  soda. 
Media  Required: 

Glucose  formate  agar. 

Glucose  formate  gelatine. 

Glucose  formate  bouillon. 

METHOD. — 

i.  Prepare  four  sets  of  cultivations: 

(A)  Oblique    glucose    formate    agar   and   incubate 
aerobically  at  37°  C. 

Oblique  glucose  formate  gelatine  and  incubate 
aerobically  at  20°  C. 

(B)  Oblique  glucose  agar  and  incubate  anaerobically 
at  37°  C. 


240  METHODS    OF    IDENTIFICATION. 

Oblique     glucose    formate    gelatine    and    incubate 
anaerobically  at  20°  C. 

(C)  Oblique    glucose    formate    agar    and   incubate 
anaerobically  at  37°  C. 

Glucose   formate   bouillon   and   incubate   anaerobi- 
cally at  37°  C. 

(D)  Oblique  glucose  formate  gelatine  and  incubate 
anaerobically  at  20°  C. 

Glucose   formate   bouillon   and   incubate   anaerobi- 
cally at  20°  C. 

2.  Seal   the  cultures  forming  set   B   in   Buchner's 
tubes  (vide  page  189). 

3.  Seal  the  cultures  forming  sets  C  and  D  in  Bulloch's 
apparatus;  exhaust  the  air  by  means  of  a  vacuum 
pump,  and  provide  for  the  absorption  of  any  residual 
oxygen  by  the  introduction  of  pyrogallic   acid  and 
caustic  soda  solution     (vide  page  194). 

4.  Observe    the    cultivations    macroscopically    and 
microscopically  at  intervals  of  twenty-four  hours  until 
the  completion,  if  necessary,  of  seven  days'  incubation. 

5.  Control  these  results. 
Gases  Other  than  Oxygen.— 

Apparatus  Required: 
Bulloch's  apparatus. 
Sterile  gas  filter  (vide  page  43). 
Gasometer  containing  the  gas  it  is  desired  to  test  or  gas 

generator  for  the    production  of  SO2,  N2O,  NO,  CO2, 

coal  gas,  etc. 

METHOD. — 

1.  Prepare  tube  cultivations  upon  the  surface   of 
solid  media  and  deposit  them  in  Bulloch's  apparatus. 

2.  Connect  up  the  inlet  tube  of  the  Bulloch's  jar 
with  the  sterile  gas  filter,  and  this  again  with  the 
delivery  tube  of  the  gasometer  or  gas  generator. 

3.  Open  both  stop-cocks  of  the  Bulloch's  apparatus 
and  pass  the  gas  through  until  it  has  completely  re- 
placed the  air  in  the  bell  jar. 


BY   PHYSICAL   METHODS.  24! 

4.  Incubate  under  optimum  conditions  as  to  tem- 
perature. 

5.  Examine  the  cultivations  at  intervals  of  twenty- 
four  hours,  until  the  completion  of  seven  days. 

6.  Remove  one  tube  from  the  interior  of  the  appa- 
ratus each  day.     If  no  growth  is  visible,  incubate  the 
tube  under   optimum   conditions   as   to   temperature 
and  atmosphere,  and  in  this  way  determine  the  length 
of  exposure  to  the  action  of  the  gas  necessary  to  kill 
the  organisms  under  observation. 

7.  Control  these  results. 
II.  Temperature. — 

(A)  Range.— 

1.  Prepare  a  series  of  ten  tube  cultivations,  in  fluid 
media,  of  optimum  reaction. 

2.  Arrange  a  series  of  incubators  at  fixed  tempera- 
ture, varying  5°  C.  and  including  temperatures  between 
5°  C.  and  50°  C. 

(Or  utilise  the  water-bath  employed  in  testing  the 
thermal  death-point  of  vegetative  forms.) 

3.  Incubate  one  tube  cultivation  of  the  organism 
aerobically  or  anaerobically,  as  may  be  necessary,  in 
each  incubator,   and  examine  at  half-hour  intervals 
for  from  five  to  eighteen  hours. 

4.  Note  that  temperature  at  which  growth  is  first 
observed  macroscopically. 

5.  Continue  the  incubation  until  the  completion  of 
seven   days.     Note   the   extremes   of   temperature  at 
which  growth  takes  place  (Range  of  temperature). 

6.  Control  these  results. 

(B)  Optimum. — 

1.  Prepare  a  second  series  of  ten  tube  cultivations 
under  similar  conditions  as  to  atmosphere  and  reaction 
of  medium. 

2.  Incubate  in  a  series  of  incubators  in  which  the 
temperature  is  regulated  at  intervals  of  i°  C.  for  five 

16 


242 


METHODS   OF   IDENTIFICATION. 


degrees  on  either  side  of  optimum  temperature  ob- 
served in  the  previous  experiment,  step  4. 

3.  Observe  again  at  half -hour  intervals  and  note 
that  temperature  at  which  growth  is  first  visible  to 
the  naked  eye  =  Optimum  temperature. 

(C)  Thermal  Death-point.— 

Moist — Vegetative  Forms: 

Apparatus  Required : 

Water-bath.  For  the  purpose  of  observing  the  thermal 
death-point  a  special  water-bath  is  necessary.  The 
temperature  of  this  piece  of  apparatus  is  controlled  by 


-^-To  water 
blower. 


Fig.  128. — Hearson's  water-bath. 

means  of  a  capsule  regulator  and  can  be  regulated  for 
intervals  of  half  a  degree  centigrade  through  a  range  of 
30°,  from  50°  C.  to  80°  C.  by  means  of  a  spring,  actuated 
by  the  handle  a,  which  increases  the  pressure  in  the 
interior  of  the  capsule.  A  hole  is  provided  for  the 
reception  of  the  nozzle  of  a  blast  pump,  so  that  a  cur- 
rent of  air  may  be  blown  through  the  water  whilst  the 
bath  is  in  use,  and  thus  ensure  a  uniform  temperature 
of  its  contents. 


BY   PHYSICAL    METHODS.  243 

Sterile  capsules. 

Flask  containing  250  c.c.  sterile  normal   saline   solution. 
Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  cubic  centi- 
metre). 
Special  loop. 
Test-tubes,  18  by  1.5  cm.,  of  thin  German  glass. 


METHOD. — 

1.  Prepare  several  tube  cultivations  on  solid  media 
of  optimum  reaction  and  incubate  for  forty-eight  hours 
under    optimum    conditions    as    to    temperature    and 
atmosphere. 

2.  Examine  preparations  from  the  cultivation  micro- 
scopically to  determine  the  absence  of  spores. 

3.  Pipette  5  c.c.  salt  solution  into  each  of  twelve 
capsules. 

4.  Suspend  three  loopfuls  of  the  growth  from  the 
surface  of  the  medium  (using  a  carefully  made  platinum 
loop  especially  reserved  for  this  purpose)  in  the  normal 
saline  solution  in  each  capsule. 

5.  Transfer  each  suspension  to  a  sterile  test-tube 
and  number  consecutively  from  i  to  12. 

6.  Adjust  the  first  tube  in  the  water-bath,  regulated 
at  40°  C.,  by  means  of  two  rubber  rings  around  the 
tube,  one  above  and  the  other  below  the  perforated 
top  of  the  bath,  so  that  the  upper  level  of  the  fluid 
in  the  tube  is  about  4  cm.  below  the  surface  of  the 
water  in  the  bath,  and  the  bottom  of  the  tube  is  a 
similar  distance  above  the  bottom  of  the  bath. 

7.  Arrange   a   control    test-tube   containing    5    c.c. 
water  under  similar  conditions.     Plug  the  tube  with 
cotton- wool  and  pass  a  thermometer  through  the  plug 
so  that  its  bulb  is  immersed  in  the  water. 

8.  Close  the  unoccupied  perforations  in  the  lid  of 
the  water-bath  by  means  of  glass  balls. 

9.  Watch  the  thermometer  in  the  test-tube  until  it 
records    a    temperature    of    40°    C.     Note    the    time. 
Ten  minutes  later  remove  the  tube  containing  the  sus- 


244  METHODS  OF  IDENTIFICATION. 

pension,    and    cool    rapidly  by  immersing  its   lower 
end  in  a  stream  of  running  water. 

10.  Pour  three  gelatine  (or  agar)  plates  containing 
respectively  0.2,  0.3,  and  0.5  c.c.  of  the  suspension, 
and  incubate. 

11.  Pipette  the  remaining  4  c.c.  of  the  suspension 
into  a  culture  flask  containing  250  c.c.   of  nutrient 
bouillon,  and  incubate. 

12.  Observe   these  cultivations  from   day  to   day. 
"No  growth"  must  not  be  recorded  as  final  until  after 
the  completion  of  seven  days'  incubation. 

13.  Extend    these    observations    to    the    remaining 
tubes  of  the  series,  but  varying  the  conditions  so  that 
each  tube  is  exposed  to  a  temperature  2°  C.  higher 
than  the  immediately  preceding  one — i.  e.,  42°  C.,  44° 
C.,  46°  C.,  and  so  on. 

14.  Note  that  temperature,  after  exposure  to  which 
no  growth  takes  place  up  to  the  end  of  seven  days' 
incubation,  =  the  thermal  death-point. 

15.  If  greater  accuracy  is  desired,  a  second  series 
of  tubes  may  be  prepared  and  exposed  for  ten  minutes 
to  fixed  temperatures  varying  only  0.5°  C.,  through 
a  range  of  5°  C.  on  either  side  of  the  previously  observed 
death-point. 

Moist — Spores :  The  thermal  death-point  in  the  case 
of  spores  is  that  time  exposure  to  a  temperature  of 
1 00°  C.  necessary  to  effect  the  death  of  all  the  spores 
present  in  a  suspension. 

It  is  determined  in  the  following  manner 

Apparatus  Required: 

Steam  can  be  fitted  with  a  delivery  tube  and  a  large  bore 

safety-valve  tube. 
Water-bath  at  100°  C. 

Brlenmeyer  flask,  500  c.c.  capacity,  containing  140  c.c. 
sterile  normal   saline   solution  and  fitted  with  rubber 
stopper  perforated  with  four  holes. 
The  rubber  stopper  is  fitted  as  follows : 

(a)  Thermometer  to  120°  C.,  its  bulb  immersed  in  the 
normal  saline. 


BY   PHYSICAL   METHODS. 


245 


(b)  Straight  entry  tube,  reaching  to  the  bottom  of 

the  flask,  the  upper  end  plugged  with  cotton- 
wool. 

(c)  Bent  syphon  tube  with  pipette,  nozzle  attached 

by  means  of   rubber   tubing  and   fitted  with 
pinch-cock. 

The  nozzle  is  protected  from  accidental  contami- 
nation by  passing  it  through  the  cotton-wool 
plug  of  a  small  test-tube. 


Fig.  129. — Apparatus  arranged  for  the  determination  of  the  death-point  of 

spores. 


(d)  A  sickle-shaped   piece  of   glass   tubing   passing 
just  through  the  stopper,  plugged  with  cotton- 
wool, to  act  as  a  vent  for  the  steam. 
Sterile  plates. 
Sterile  pipettes. 

Sterile  test-tubes  graduated  to  5  c.c. 
Media  Required: 
Gelatine  or  agar. 
Culture  flasks  containing  200  c.c.  nutrient  bouillon. 


246  METHODS  OF  IDENTIFICATION. 

METHOD. — 

1.  Prepare  twelve  tube  cultivations  upon  the  sur- 
face (or  two  cultures    in  large  flat  culture  bottles— 
-vide   page   19)   of    nutrient  agar  and  incubate  under 
the  optimum  conditions  (previously  determined),  for 
the  formation  of  spores. 

Examine    preparations    from    the    cultures    micro- 
scopically to  determine  the  presence  of  spores. 

2.  Pipette  5  c.c.  sterile  normal  saline  into  each  cul- 
ture tube  or  30  c.c.  into  each  bottle  and  by  means  of  a 
sterile  platinum  spatula  emulsify  the  entire  surface 
growth  with  the  solution. 

3.  Add  the  60  c.c.  emulsion  to  140  c.c.  normal  saline 
contained  in  the  fitted  Erlenmeyer  flask. 

4.  Place  the  flask  in  the  water-bath  of  boiling  water. 

5.  Connect  up  the  straight  tube,  after  removing  the 
cotton- wool  plug, with  the  delivery  tube  of  the  steam 
can;  remove  the  plug  from  the  vent  tube. 

6.  When  the  thermometer  reaches  100°  C.,  syphon 
off   5  c.c.    of  the   suspension  into   the  sterile  gradu- 
ated test-tube  and  pour  plates  and  prepare  flask  cul- 
tures as  in  the  previous  experiments. 

7.  Repeat   this  process  at  intervals  of   twenty-five 
minutes'  steaming. 

8.  Control  these  experiments,  but  in  this  instance 
syphon  off  portions  of  the  suspension  at  intervals  of 
one-half  or  one  minute  during  the  five  or  ten  minutes 
preceding  the  previously  determined  death-point. 

Thermal  Death-point. — 
Dry — Vegetative  Forms: 

Apparatus  Required: 

Hot-air  oven,  provided  with  thermo-regulator. 
Sterile  cover-slips. 

Flask  containing  250  c.c.  sterile  normal  saline  solution. 
Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  cubic  centi- 
metre). 

Case  of  sterile  capsules. 
Crucible  tongs. 


BY  PHYSICAL  METHODS.  247 

METHOD.— 

1.  Prepare  an  emulsion  with  three  loopfuls  from  an 
optimum  cultivation  in  5  c.c.  normal  saline  in  a  ster- 
ile capsule  and  examine  microscopically  to  determine 
the  absence  of  spore  forms. 

2.  Make  twelve  cover-slip  films  on  sterile  cover-slips; 
place  each  in  a  sterile  capsule  to  dry. 

3.  Expose  each  capsule  in  turn  in  the  hot-air  oven 
for  ten  minutes  to  a  different  fixed  temperature,  vary- 
ing 5°  C.  between  60°  C.  and  120°  C. 

4.  Remove  each  capsule  from  the  oven  with  crucible 
tongs  immediately   the  ten  minutes   are  completed; 
remove  the  cover-glass  from  its  interior  with  a  sterile 
pair  of  forceps. 

5.  Deposit  the  film  in  a  flask  containing  200  c.c. 
nutrient  bouillon. 

6.  Prepare  subcultivations  from  such  flasks  as  show 
evidence  of  growth, to  determine  that  no  contamination 
has  taken  place. 

7.  Control  the  result  of  these  experiments. 
Dry — Spores : 

Apparatus  Required: 
As  for  vegetative  forms. 

METHOD.— 

1.  Prepare  an  oblique  agar  tube  cultivation  and  in- 
cubate under  optimum  conditions  as  to  spore  forma- 
tions. 

2.  Pipette  5  c.c.  sterile  normal  saline  into  the  culture 
tube  and  emulsify  the  entire  surface  growth  in  it.     Ex- 
amine microscopically  to  determine  the  presence  of 
spores  in  large  numbers. 

3.  Spread  thin  even  films  on  twelve  sterile  cover- 
slips  and  place  each  cover-slip  in  a  separate  sterile 
capsule. 

4.  Expose  each  capsule  in  turn  for  ten  minutes  to  a 
different  fixed  temperature,   varying  5°  C.,  between 
100°  C.  and  160°  C. 


248  METHODS    OF    IDENTIFICATION. 

5.  Complete  the  examination  as  for  vegetative  forms. 
III.  Reaction  of  Medium.— 

(A)  Range. — 

1 .  Prepare  a  bouillon  culture  of  the  organism  and  in- 
cubate, under  optimum  conditions  as  to  temperature 
and  atmosphere,  for  twenty-four  hours. 

2.  Pipette  o.i  c.c.  of  the  cultivation  into  a  sterile 
capsule;    add  9.9  c.c.  sterile  bouillon  and  mix  thor- 
oughly. 

3.  Prepare  a  series  of  tubes  of  nutrient  bouillon  of 
varying  reactions,  from  +25  to  — 30  (vide  page  133), 
viz.:   +25,   +20,   +15,   +10,   +5,  neutral, — 5,  — 10, 
—15,  —20,  —25,  —30. 

4.  Inoculate   each   of   the   bouillon  tubes   with  o.i 
c.c.  of  the  diluted  cultivation  and  incubate  under  opti- 
mum conditions. 

5.  Observe    the    cultures    at    half -hourly    intervals 
from  the  third  to  the  twelfth  hours.     Note  the  reaction 
of  the  tube  or  tubes  in  which  growth  is  first  visible 
macroscopically  (probably  optimum  reaction). 

6.  Continue  the  incubation  until  the  completion  of 
forty-eight  hours.     Note  the  extremes  of   acidity  and 
alkalinity  in  which  macroscopical  growth  has  developed 
(Range  of  reaction) . 

7.  Control  the  result  of  these  observations. 

(B)  Optimum  Reaction. — The  optimum  reaction  has 
already  been  roughly  determined  whilst  observing  the 
range.     It   can  be   fixed   within   narrower  limits   by 
inoculating  in  a  similar  manner  a  series  of  tubes  of 
bouillon  which  have  a  smaller  variation  in  reaction  than 
those  previously  employed  (say,  i  instead  of  5)  for  five 
points  on  either  side  of  the  previously  observed  opti- 
mum.    For  example,  optimum  reaction  in  the  set  of 
experiments  to  determine  the  range  was  +  10.      Now 
plant  tubes  having  reactions  of  +15,  +14,  +13,  +12, 
+  11,    +10,   +9,   +8,   +  7,   +6,   +5,  and  observe  as 
before. 


BY   PHYSICAL    METHODS.  249 

IV.  Resistance  to  Lethal  Agents. — 

(A)  Desiccation. — 

Apparatus  Required: 

Miiller's  desiccator.  This  consists  of  a  bell  glass  fitted 
with  an  exhaust  tube  and  stop-cock  (d),  which  can  be 
secured  to  a  plate-glass  base  (c)  by  means  of  wax  or 
grease.  It  contains  a  cylindrical  vessel  of  porous  clay 
(a)  into  which  pure  sulphuric  acid  is  poured  whilst  the 
material  to  be  dried  is  placed  within  its  walls  on  a 
glass  shelf  (b).  The  air  is  exhausted  from  the  interior 
and  the  acid  rapidly  converts  the  clay  vessel  into  a 
large  absorbing  surface. 


^  <* 

Fig.  130. — Muller's  desiccator. 

Exhaust  pump. 

Pure  concentrated  sulphuric  acid. 

Sterile  cover-slips. 

Sterile  forceps. 

Culture  flask  containing  200  c.c.  nutrient  bouillon. 

Sterile  ventilated  Petri  dish.  This  is  prepared  by  bending 
three  short  pieces  of  aluminium  wire  into  V  shape  and 
hanging  these  on  the  edge  of  the  lower  dish  and  resting 
the  lid  upon  them  (Fig.  131). 


250  METHODS  OF  IDENTIFICATION. 

METHOD. — 

1.  Prepare  a  surface  cultivation  on  nutrient  agar  in 
a  culture  bottle  and  incubate  under  optimum  condi- 
tions for  forty-eight  hours. 

2.  Examine    preparations     from     the     cultivation, 
microscopically,  to  determine  the  absence  of  spores. 

3.  Pipette  5  c.c.  sterile  nor- 
mal saline  solution  into  the 
flask  and  suspend  the  entire 
growth  in  it. 

4-  Spread  the  suspension  in 
thin,  even  films  on  sterile 
cover-slips  and  deposit  inside  sterile  " plates"  to  dry. 

5.  As  soon  as  dry,  transfer  the  cover-slip  films  to  the 
ventilated  Petri  dish  by  means  of  sterile  forceps. 

6.  Place  the  Petri  dish  inside  the  M tiller's  desiccator; 
fill  the  upper  chamber  with  pure  sulphuric  acid,  cover 
with  the  bell  jar,  and  exhaust  the  air  from  its  interior. 

7.  At  intervals  of  five  hours  admit  air  to  the  appara- 
tus, remove  one  of  the  cover-slip  films  from  the  Petri 
dish,  and  transfer  it  to  the  interior  of  a  culture  flask, 
with   every   precaution   against   contamination.     Re- 
seal  the  desiccator  and  exhaust. 

8.  Incubate  the  culture  flask  under  optimum  condi- 
tions until  the  completion  of  seven  days,  if  necessary. 

9.  Pour  plates  from  those  culture  flasks  which  grow, 
to  determine  the  absence  of  contamination. 

10.  Repeat  these  observations  at  hourly  intervals  for 
the  five  hours  preceding  and  succeeding  the  death 
time,  as  determined  in  the  first  set  of  experiments. 

(B)  Light.— 

(a)  Diffuse  Daylight: 

1.  Prepare  a  tube  cultivation  in  nutrient  bouillon, 
and  incubate  under  optimum  conditions,  for  forty-eight 
hours. 

2.  Pour  twenty  plate  cultivations,  ten  of  nutrient 


BY    PHYSICAL    METHODS.  251 

gelatine  and  ten  of  nutrient  agar,  each  containing  o.i 
c.c.  of  the  bouillon  culture. 

3.  Place  one  agar  plate  and  one  gelatine  plate  into 
the  hot  and  cold  incubators,  respectively,  as  controls. 

4.  Fasten  a  piece  of  black  paper,  cut  the  shape  of  a 
cross  or  star,  on  the  centre  of  the  cover  of  each  of  the 
remaining  plates  (Fig.  132). 

5.  Expose  these  plates  to  the  action  of  diffuse  day- 
light (not  direct  sunlight)  in  the  laboratory  for  one, 
two,  three,  four,  five, 

six,  eight,  ten,  twelve 
hours. 

6.  After    exposure 
to  light,  incubate  un- 
der optimum   condi- 
tions. 

7.  Examine      the 
plate  cultivations  af- 

.  1         Fig.  132. — Plate  with  star  for  testing  effect 

ter    twenty-four  and  Of  iight. 

forty-eight  hours'  in- 
cubation, and  compare  with  the  two  controls.     Record 
results.     If  growth  is  absent  from  that  portion  of  the 
plate  unprotected  by  the  black  paper,  continue  the  incu- 
bation and  daily  observation  until  the  end  of  seven  days. 

8.  Control  the  results. 
(b)  Direct  Sunlight: 

1.  Prepare   plate   cultivations   precisely   as   in   the 
former  experiments  and  place  the  two  controls  in  the 
incubators. 

2.  Arrange  the  remaining  plates  upon  a  platform  in 
the  direct  rays  of  the  sun. 

3.  On  the  top  of  each  plate  stand  a  small  glass  dish 
14  cm.  in  diameter  and  5  cm.  deep. 

4.  Fill  a  solution  of  potash  alum  (2  per  cent,  in  dis- 
tilled water)  into  each  dish  to  the  depth  of  2  cm.  to 
absorb  the  heat  of  the  sun's  rays  and  so  eliminate  possi- 
ble effects  of  temperature  on  the  cultivations. 


252  METHODS    OF    IDENTIFICATION. 

5.  After  exposures  for  periods  similar  to  those  em- 
ployed in  the  preceding  experiment,  incubate  and  com- 
plete the  observation  as  above. 

(c)  Primary  Colours:  Each  colour — violet,  blue, 
green,  red,  and  yellow — must  be  tested  separately. 

1.  Prepare  plate   cultivations,   as   in   the  previous 
"light"  experiments,  and  incubate  controls. 

2.  Fasten  a  strip  of  black  paper,  3  cm.  wide,  across 
one  diameter  of  the  cover  of  each  plate. 

3.  Coat  the  remainder  of  the  surface  of  the  cover 
with  a  film  of  pure  photographic  collodion  which  con- 
tains 2  per  cent,  of  either  of  the  following  aniline  dyes, 
as  may  be  necessary: 

Chrysoidin  (for  red) . 
Aurantia  (for  orange). 
Naples  yellow  (for  yellow). 
Malachite  green  (for  green). 
Eosin,  bluish  (for  blue). 
Methyl  violet  (for  violet). 

4.  Expose  the  plates,  thus  prepared,  to  bright  day- 
light (but  not  direct  sunlight)  for  varying  periods,  and 
complete  the  observations  as  in  the  preceding  experi- 
ments. 

5.  Control  the  results. 

(C)  Heat. — (Vide  Thermal  Death-point,  page  242.) 

(D)  Antiseptics    and    Disinfectants. — (Vide    Testing 
Germicides,  page  359.)     Testing  the  organism  under 
observation  against,  for  example, 

Bichloride  of  mercury; 

Formaldehyde ; 

Carbolic  acid; 

noting  (a)  strength  of  solution;    (b)  duration  of  ex- 
posure necessary  to  produce  death. 

The  Agglutination  Reaction. — This  test,  which  is 
variously  known  as  the  agglutination  reaction,  clump- 
ing reaction,  or  Gruber's  reaction,  depends  upon  the  fact 
that  the  blood-serum  of  an  animal  immunised  against 


BY   PHYSICAL   METHODS.  253 

a  certain  micro-organism  possesses  the  power  of  agglu- 
tinating (or  collecting  together  in  clumps  and  masses) 
watery  suspensions  of  that  particular  microbe.  It  was 
first  applied  by  Durham  and  Gruber  in  the  identifica- 
tion of  races  of  cholera  vibrios,  and  was  afterwards 
extended  by  various  workers  to  other  species  of  bac- 
teria, such  as  B.  typhosus,  B.  pestis,  M.  melitensis, 
etc. 

The  converse  of  the  test — viz.,  the  diagnosis  of  dis- 
ease by  the  determination  of  the  particular  pathogenic 
organism  that  is  agglutinated  by  the  blood-serum  of 
the  patient — is  often  termed  Widal's  reaction,  and  is 
extensively  used  in  the  diagnosis  of  enteric  fever,  when 
the  serum  from  a  suspected  case  of  typhoid  is  tested 
against  a  bouillon  cultivation  of  an  authentic  Bacillus 
typhosus. 

It  is  now  generally  agreed  that  the  reaction  is  unre- 
liable unless  performed  under  certain  conditions. 

1.  As  to  the  period  of  time  the  suspension  of  the 
organism  is  in  contact  with  specific  serum.     This  must 
not  exceed  thirty  minutes. 

2.  As  to  the  strength  of  the  solution  of  specific  serum 
employed  in  the  test.    This  must  not  exceed  5  per  cent. 

The  method  of  employing  the  test  and  the  prepara- 
tion of  the  serum  solution  are  best  considered  sepa- 
rately, and  the  test  itself  illustrated  by  an  example  such 
as  the  confirmation  of  the  identity  of  a  bacillus  pro- 
visionally regarded  as  the  B.  typhosus. 

(A)  Preparation.— 

Collection  of  the  Specific  Serum: 

Apparatus  Required: 
Razor. 
Liquid  soap. 
Cotton-wool. 

Two  per  cent,  lysol  solution  in  drop  bottle. 
Absolute  alcohol  in  drop  bottle. 
Hare-lip  pin  or  pointed  scalpel. 
Blood  pipette  (vide  page  22). 


254  METHODS  OF  IDENTIFICATION. 

METHOD. — 

1 .  Select  a  rabbit  which  has  been  immunised  to  the  B. 
typhi  abdominalis,  and  have  it  firmly  held  by  an  assist- 
ant. 

2.  Shave  the  dorsal  surface  of  the  ear,  over  the  poste- 
rior auricular  vein. 

NOTE. — The  serum  may  also  be  obtained  from  the 
lobe  of  the  ear  of  a  patient  suffering  or  convalescent 
from  enteric  fever,  by  carrying  out  the  succeeding 
steps  (see  Fig.  133). 

3.  Sterilise  the  skin  by  washing  with  lysol. 

The  lysol  should  be  applied  with  sterile  cotton-wool 


Fig.  133. — Collecting  blood. 

and  the  ear  vigourously  rubbed,  not  only  to  remove 
superficial  scales  of  epithelium,  but  also  to  render  the 
ear  hyperaemic  and  the  vein  prominent. 

4.  Remove  the  lysol  with  absolute  alcohol. 

5.  Dry  the  sterilised  area  of  skin  with  sterile  cotton- 
wool. 

6.  Puncture  the  vein  with  the  sterile  hare-lip  pin 
and  collect  the  issuing  blood  in  the  blood  pipette,  thus : 

Hold  one  of  the  narrow  .tube-ends  in  contact  with 
the  blood  and  depress  the  other  end.  The  blood  will 
run  into  the  pipette  by  gravity.  When  the  tube  is  full 
to  the  shoulder,  remove  the  pipette,  place  the  clean  end 


BY   PHYSICAL,   METHODS. 


255 


to  the  lips,  and  aspirate  gently,  so  that  the  blood  flows 
into  the  barrel  of  the  pipette. 

Hold  the  pipette  horizontally,  and  seal  the  ends  in  a 
Bunsen  flame. 

Rest  the  pipette  in  the  horizontal  position,  by  its 
ends  on  the  rim  of  a  tumbler  or  beaker,  so  that  its  bar- 
rel is  suspended,  and  allow  the  blood  to  coagulate 
(Fig.  134,  a).  This  will  probably  take  about  twenty 
to  thirty  minutes. 

Place  the  pipette  in  the  vertical  position,  clean  end 
downwards,  in  a  beaker  or  wire  stand,  and  set  it  in 


Fig.  134. — Collecting  serum  :  a,  formation  of  clot ;  l>,  separation  of  serum. 


the  ice-chest  for  an  hour  or  so,  for  the  clear  serum  to 
separate  and  collect  in  the  clean  end  of  the  pipette 
(Fig.  134,  b). 

Dilution  of  the  Specific  Serum: 

Apparatus  Required: 

Sterile  graduated  capillary  pipettes  (to  contain  10  c.mm.). 

Sterile  graduated  capillary  pipettes  (to  contain  90  c.mm.). 

Small  sterile  test-tubes  (5  cm.  by  0.5  cm.). 

Tube  of  nutrient  bouillon. 

Pipette  of  specific  serum. 

Three-square  file. 

Glass  capsule,  nearly  full  of  dry  silver  sand. 

Grease  pencil. 


256  METHODS  OF  IDENTIFICATION. 

METHOD. — 

1.  Take  three  sterile  test-tubes  and  number  them  i, 
2,  and  3. 

2.  Pipette  90  c.mm.  sterile  bouillon  into  each  tube, 
and  stand  it  upright  in  the  sand  in  the  capsule. 

3.  Make  a  file  scratch  on  the  blood  pipette  above  the 
upper  level  of  .the  clear  serum,  and  snap  off  the  narrow 
tube  containing  the  serum. 

4.  Remove  10  c.mm.  of  the  serum  from  the  blood 
pipette  tube,  and  mix  it  thoroughly  with  the  bouillon 
in  tube  No.  i ;  =  specific  serum  solution,  10  per  cent. 

5.  Remove  10  c.mm.  of  the  solution  from  tube  No.  i 
by  means  of  a  fresh  pipette,  and  mix  it  with  the  con- 
tents of  tube  No.  2;  =  specific  serum  solution,  i  per 
cent. 

6.  Remove  10  c.mm.  of  the  solution  from  tube  No.  2 
by  means  of  a  fresh  pipette,  and  mix  it  with  the  con- 
tents of  tube  No.  3 ;  —  specific  serum  solution,  o.  i  per 
cent. 

(B)  Application. — 

The  Microscopical  Reaction: 

Apparatus  Required: 

Five  hanging-drop  slides  (or  preferably  two  slides,  with 

two  cells  mounted  side  by  side   on   each  (Fig.  47,  a), 

and  one  slide  with  one  cell  only). 
Vaseline. 
Cover-slips. 
Platinum  loop. 
Grease  pencil. 
Eighteen-  to  twenty-four-hour-old  bouillon  cultivation  of 

the  organism  to  be  tested  (Bacillus  typhi  abdominalis?). 
Pipette  end  with  the  remainder  of  the  undiluted  serum. 
Tubes  containing  the  three  solutions  of  the  specific  serum, 

10,  i,  and  o.i  per  cent,  respectively. 

METHOD. — 

i.  Make  five  hanging-drop  preparations,  thus: 
(a)  One  loopful  of  bouillon  cultivation  -f  one  loopful 
sterile  bouillon;  label  "Control." 


BY   PHYSICAL    METHODS.  257 

(b)  One   loopful   culture  +    one   loopful  undiluted 
serum;  label  50  per  cent. 

Mount  these  two  cover-slips  on  a  double-celled  slide. 

(c)  One  loopful  bouillon  culture   +  one  loopful  10 
per  cent,  serum;  label  5  per  cent. 

Mount  this  on  single-cell  slide. 

(d)  One  loopful  bouillon  culture  +  one  loopful   i 
per  cent,  serum;  label  0.5  per  cent. 

(e)  One  loopful  bouillon  culture  +  one  loopful  o.i 
per  cent,  serum;  label  0.05  per  cent. 

Mount  these  two  cover-slips  on  a  double-celled 
slide. 

2.  Note  the  time:  Examine  the  control  to  deter- 
mine that  the  bacilli  are  motile  and  uniformly  scattered 
over  the  field — not  collected  into  masses. 

3.  Next  examine  the  50  per  cent,  preparation. 

If  the  test  is  giving  a  positive  reaction,  the  bacilli 
•will  be  collected  in  large  clumps. 

If  the  test  is  giving  a  negative  reaction,  the  bacilli 
'may  be  collected  in  large  clumps. 

4.  Observe  the  5  per  cent,  preparation  microscopi- 
cally. 

If  the  bacilli  are  aggregated  into  clumps,  positive  re- 
action. 

If  the  bacilli  are  not  aggregated  into  clumps,  observe 
until  thirty  minutes  from  the  time  of  preparation  be- 
fore recording  a  negative  reaction. 

5.  Examine  the  0.5  and  0.05  per  cent,  preparations^ 
These  may  or  may  not  show  agglutination  when  the- 
result  of  the  examination  of  the  5  per  cent,  prepara- 
tion is  positive,  according  to  the  potency  of  the  specific: 
serum;  and  by  dilutions  such  as  these  a  quantitative: 
comparison  of  the  valency  of  specific  sera  may  be  ob- 
tained. 

NOTE. — The     graduated     pipettes     supplied     with 
Thoma's  haematocytometer  (intended  for  the  collection 
of  the  specimen  of  blood  required  for  the  enumeration 
17 


258  METHODS    OF    IDENTIFICATION. 

of  leucocytes),  giving  a  dilution  of  i  in  10, — i.  e.,  10  per 
cent., — may  be  substituted  for  the  graduated  capillary 
pipettes  referred  to  above,  if  the  vessel  in  which  the 
serum  has  been  separated  is  of  sufficiently  large  diam- 
eter to  permit  of  their  use. 

A  handy,  though  somewhat  crude,  method  of  apply- 
ing this  microscopical  test  is  carried  out  as  follows: 

Apparatus  Required: 

Pipette  containing  immune  serum. 

Eighteen-  to  twenty-four-hour-old  broth  cultivation  of  the 

organism  to  be  tested. 
Tube  of  sterile  broth  (or  sterile  water). 
Cover-slips. 
Platinum  loop. 
Hanging-drop  slides. 
Vaseline. 
Grease  pencil. 

METHOD. — 

1.  Flame  a  clean  cover-slip  and  rest  it  on  a  piece  of 
blotting  paper. 

2.  By  means  of  a  sterile  platinum  loop  place  nine 
as  nearly  as   possible   equal  loopfuls  of  sterile   broth 
(or  sterile  water)  on  the  surface  of  the  cover-slip. 

3.  Sterilise  the  loop,  fill  it  once  with  the  serum  to 
be  tested,  and  mix  thoroughly  with  the  nine  drops 
of  diluent.     This  gives  approximately  a  10  per  cent, 
solution  of  serum. 

4.  Flame  a  second  clean  cover-slip  and  place  it  by 
the  side  of  the  first. 

5.  Sterilise  the  loop,  charge  it  with  the  serum  solu- 
tion, and  deposit  a  drop  on  the  surface  of  the  second 
cover-slip. 

6.  Sterilise  the  loop,  and  add  a  loopful  of  the  broth 
cultivation  to  the  drop  of  diluted  serum,   and  mix 
thoroughly. 

7.  Mount  the  cover-slip  as  a  hanging  drop,  label 
5  per  cent.,  and  examine  microscopically. 

Higher  dilutions  can  be  prepared  in  a  similar  man- 
ner. 


PATHOGENESIS.  259 

The  Macroscopical  Reaction : 

Apparatus  Required: 

Sterile  graduated  capillary  pipettes  to  contain  90  c.mm. 

Eighteen-  to  twenty-four-hour-old  bouillon  cultivation  of 
the  organism  to  be  tested. 

Three  test-tubes  containing  the  10,  i,  and  o.i  per  cent,  so- 
lutions of  specific  serum  (about  90  c.mm.  remaining  in 
each). 

Sedimentation  tubes  ('vufepage  23). 

METHOD.— 

1.  Pipette  90  c.mm.  of  the  bouillon  culture  into  each 
of  the  tubes  containing  the  diluted  serum. 

2.  Fill  a  sedimentation  tube   (by  aspirating)  from 
the  contents  of  each  tube.     Seal  off  the  lower  ends  of 
the  sedimentation  tubes  in  the  Bunsen  flame. 

3.  Label  each  tube  with  the  dilution  of  serum  that  it 
contains — viz.,  5,  0.5,  and  0.05  per  cent. 

4.  Place  the  pipettes  in  a  vertical  position,   in  a 
beaker,  in  the  incubator  at  37°  C.,  for  one  or  two 
hours. 

5.  Observe  the  granular  precipitate  which  is  thrown 
down  when  the  reaction  is  positive,  and  the  uniform 
turbidity  of  the  negative  reaction. 

PATHOGENESIS. 

i.  Living  Bacteria. — 

(a)  Psychrophilic  Bacteria:  When  the  organism  will 
only  grow  at  or  below  18°  to  20°  C., 

1.  Prepare  cultivations  in  nutrient  broth  and  in- 
cubate under  optimum  conditions. 

2.  After  seven  days'  incubation  inject  that  amount 
of  the  culture  corresponding  to    i    per  cent,   of  the 
body- weight  of  a  selected  frog,  into  its  dorsal  lymph 
sac. 

3.  Observe  until  death  takes  place,  or,  in  the  event 
of  a  negative  result,  until  the  completion  of  twenty- 
eight  days. 

4.  If   death  .occurs,   make    a    careful   post-mortem 
examination  (vide  page  287). 


260  METHODS    OF    IDENTIFICATION. 

(b)  Mesophilic  Bacteria:  When  the  organism  grows 
at  35°  to  37°  C., 

1 .  Prepare  cultivations  in  nutrient  broth  and  incu- 
bate under  optimum  conditions  for  forty-eight  hours. 

2.  Inoculate  a  selected  white  mouse,  subcutaneously 
at  the  root -of  the  tail,  with  an  amount  of  cultivation 
equivalent  to  i  per  cent,  of  its  body-weight. 

3.  Inoculate  a  second  mouse  intraperitoneally  with 
a  similar  dose. 

4.  Observe  carefully  until  death  occurs,  or  until  the 
lapse  of  twenty-eight  days. 

5.  If  the  inoculated  animals  succumb,  make  com- 
plete post-mortem  examination. 

If  death  follows  shortly  after  the  injection  of  cul- 
tivations of  bacteria,  the  inoculation  experiments 
should  be  repeated  two  or  three  times.  Then,  if  the 
organism  under  observation  invariably  exhibits  patho- 
genic effects,  steps  should  be  taken  to  ascertain,  if 
possible,  the  minimal  lethal  dose  (as  described  on 
page  269)  of  the  growth  upon  solid  media  for  the  frog 
or  white  mouse  respectively.  Other  experimental 
animals — e.  g.,  the  white  rat,  guinea-pig,  and  rabbit — 
should  next  be  tested  in  a  similar  manner. 

2.  Toxins. — Prepare  cultivations  of  the  organism 
under  observation  in  glucose  formate  broth,  and  in- 
cubate for  fourteen  days  under  optimum  conditions. 

(a)  Intracellular  or  Insoluble  Toxins: 

1.  Heat  the  fluid  culture  in  a  water-bath  at  60°  C. 
for  twenty  minutes.      (The  resulting  sterile,   turbid 
fluid  is  often  spoken  of  as  "  killed  "  culture.) 

2.  Inject  subcutaneously  that  amount  of  the  culti- 
vation corresponding  to  i  per  cent,  of  the  body- weight 
of  the  selected  animal,  usually  one  of  the  small  rodents. 

3.  Inoculate  a  tube  of  sterile  bouillon  with  a  similar 
quantity,   and  inoculate  under  optimum  conditions. 
This  "  control"  then  serves  to  demonstrate  the  freedom 
of  the  toxin  from  living  bacteria. 


PATHOGENESIS. 


26l 


4.  Observe  during  life  or  until  the  completion  of 
twenty-eight  days,  and  in  the  event  of  death  occurring 
during  that  period,  make  a  complete  post-mortem  ex- 
amination. 

5.  Repeat  the  experiment   at  least  once.     In   the 
event  of  a  positive  result  estimate  the  minimal  lethal 
dose  of  "killed"   culture  for  each  of  the  species   of 
animals  experimented  upon. 

(b)  Extracellular  or  Soluble  Toxins: 

i.  Filter  the  cultivation  through  a  porcelain  filter 
candle  (Berkfeld)  into  a  sterile  filter  flask,  arranging 
the  apparatus  as  in  the  accompanying  figure  (Fig.  135). 


Fig.  135. — Apparatus  arranged  for  toxin  filtration. 

2.  Inoculate   mice,   rats,   guinea-pigs,    and   rabbits 
subcutaneously   with   that   quantity   of   toxin   corre- 
sponding to  i  per  cent,  of  the  body-weight  of  each 
respectively,  and  observe,  if  necessary,  until  the  com- 
pletion of  one  month. 

3.  Inoculate  a  "control"  tube  of  bouillon  with  a 
similar  quantity  and  incubate. 

4.  In  the  event  of  a  fatal  termination  make  com- 
plete and  careful  post-mortem  examinations. 

5.  Repeat  the  experiments  and,  if  the  results  are 
positive,  ascertain  the  minimal  lethal  dose  of  toxin 
for  each  of  the  susceptible  animals. 


XV.  EXPERIMENTAL  INOCULATION  OF 
ANIMALS. 

THE  animals  generally  employed  in  the  study  of 
the  pathogenic  properties  of  the  various  micro-organ- 
isms are: 

Mouse. 
Rat. 

Guinea-pig. 
Rabbit. 
Pigeon. 
Fowl. 

Preparation. — Before  inoculation,  the  experimental 
animals  should  be  carefully  examined,  to  avoid  the 
risk  of  employing  diseased  animals;  the  weight  should 
be  recorded  and  the  rectal  temperature  taken. 

Weighing. — The  larger 
animals  are  most  conve- 
niently weighed  in  a  deci- 
mal scale  provided  with  a 
metal  cage  for  their  re- 
ception instead  of  the 
ordinary  pan  (Fig.  136). 


Mice  and  rats  are  weighed 
in  a  modification  of  the 
letter  balance,  weighing 
to  250  grammes,  which 
has  a  conical  wire  cage 
substituted  for  its  pan 

(Fig-  137). 

The  weight  of  inoculated  animals  should  be  observed 
and  recorded  each  day,  at  precisely  the  same  hour, 
during  the  entire  period  of  observation,  preferably 
before  the  morning  feeding. 

262 


Fig.   ^6. — Rabbit  scales. 


CAGES. 


263 


Temperature. — To  take  the  rectal  temperature  of 
any  of  the  laboratory  animals,  the  animal  should  be 
firmly  held  by  an  assistant  and  the  bulb  of  an  ordinary 
clinical  thermometer,  well  greased  with  vaseline,  intro- 
duced just  within  the  sphincter  ani.  Allow  it  to  remain 
in  this  position  for  a  few  seconds,  and  then  push  it 
on  gently  and  steadily 
until  the  entire  bulb 
and  part  of  the  stem, 
as  far  as  the  constric- 
tion, have  passed  into 
the  rectum.  Three  to 
five  minutes  later,  the 
time  varying,  of  course, 
with  the  sensibility  of 
the  thermometer  used, 
withdraw  the  instru- 
ment and  take  the 
reading. 

Daily,  if  not  more 
frequently,  observa- 
tions should  be  made 
of  the  temperature 
of  inoculated  animals 
during  the  entire  pe- 
riod they  are  under 
observation. 

Cages. — During  the  period  which  elapses  between 
inoculation  and  death,  or  complete  recovery,  the 
experimental  animals  must  be  kept  in  suitable  re- 
ceptacles that  can  easily  be  kept  clean  and  which  can 
be  readily  disinfected. 

The  mouse  is  usually  stored  in  a  glass  jar  (Fig.  138) 
ii  cm.  high  and  n  cm.  in  diameter,  closed  by  a  wire 
gauze  top  which  is  weighted  with  lead  or  fastened  to 
the  mouth  of  the  jar  by  a  bayonet  catch.  A  matter 
of  great  convenience  is  a  small  oblong  label  5  cm.  by 


J37' — Mouse  scales. 


264    EXPERIMENTAL  INOCULATION  OF  ANIMALS. 


2.5  cm.,  sand-blasted  on  the  side  of  the  cylinder,  as 
marks  made  upon  this  with  an  ordinary  lead  pencil 

show  up  well  and  only  require 
the  use  of  a  damp  cloth  to 
remove  them  (Fig.  138). 

The  rat  is  kept  under  ob- 
servation in  a  glass  jar  simi- 
lar, but  larger,  to  that  used 
for  the  mouse. 

These  jars  are  sterilised 
after  use  either  by  chemical 
reagents  or  by  autoclaving. 

The  rabbit  and  the  guinea- 
pig  are  confined  in  cages  of 
suitable  size,  made  entirely  of 
metal  (Fig.  139).  The  sides 
and  top  and  bottom  are  of  woven  wire  .work ;  beneath 
the  cage  is  a  movable  metal  tray  filled  with  sawdust, 
for  the  reception  of  the  excreta.  The  cage  as  a  whole 
is  raised  from  the  ground  on  short  legs.  The  sides, 


Fig.  138. — Mouse  jar. 


Fig.  139. — Metal  rabbit  cage. 

etc.,  are  generally  hinged  so  that  the  cage  packs  up 

flat,  for  convenience  of  storing  and  also  of  sterilising. 

The    ordinary   rat   cage,  a   rectangular    wire- work 


APPARATUS   REQUIRED.  265 

box,  30  cm.  from  front  to  back,  20  cm.  wide,  and  14 
cm.  high,  makes  an  excellent  cage  for  guinea-pigs  if 
fitted  with  a  shallow  zinc  tray, 35  by  24  cm., for  it  to 
stand  upon. 

These  cages  are  sterilised  after  use  either  by  auto- 
claving  or  spraying  with  formalin. 

The  tray  which  receives  the  dejecta  should  be 
cleaned  out  and  supplied  with  fresh  sawdust  each  day, 
and  the  soiled  sawdust,  remains  of  food,  etc.,  should 
be  cremated. 

As  animal  inoculation  is  purely  a  surgical  operation, 
the  necessary  instruments  will  be  similar  to  those  em- 
ployed by  the  surgeon,  and,  like  them,  must  be  sterile. 
In  the  performance  of  the  inoculation  strict  attention 
must  be  paid  to  asepsis  and  suitable  precautions 
adopted  to  guard  against  accidental  contamination  of 
the  material  to  be  introduced  into  the  animal.  In 
addition,  the  hands  of  the  operator  should  be  care- 
fully disinfected. 

The  list  of  apparatus  used  in  animal  inocula- 
tions given  below  comprises  practically  everything 
needed  for  any  inoculation.  Needless  to  remark,  all 
the  apparatus  will  never  be  required  for  any  one 
inoculation. 

Apparatus  Required  for  Animal  Inoculation: 

i.  Water  steriliser  (vide  page  38).  It  is  also  convenient 
to  have  a  second  water  steriliser,  similar  but  smaller 
(23  by  7  by  5  cm.),  for  the  sterilisation  of  the  syringes. 


Fig.   140. — Hypodermic  syringe  with  finger  rests. 

2.  Injecting  syringe.  The  best  form  is  one  of  the  ordi- 
nary hypodermic  pattern,  fitted  with  finger  rests, 
but  with  the  leather  washers  and  the  packing  of  the 
piston  replaced  by  those  made  of  asbestos  (Fig.  140). 
The  instrument  must  be  easily  taken  to  pieces,  and 


266      EXPERIMENTAL  INOCULATION  OF  ANIMALS. 

spare  parts  should  be  kept  on  hand  to  replace  acci- 
dental breakage  or  loss.  A  good  supply  of  needles 
must  be  kept  on  hand,  both  sharp-pointed  and  with 
blunt  ends.  To  sterilise  the  syringe,  fill  it  with  water, 
loosen  the  packing  of  the  piston  and  all  the  screw 
joints,  place  it  in  the  steriliser  and  boil  for  at  least 
five  minutes.  Disinfect  the  syringe  after  use,  in  a 
similar  manner.  The  needles,  which  are  exceedingly 
apt  to  rust  after  being  boiled,  should  be  stored  in 
a  pot  of  absolute  alcohol  when  not  in  use. 

3.  Surgical  instruments,  such  as 

Scissors,  probe  and  sharp-pointed. 

Dissecting  forceps  of  various  patterns. 

Pressure  forceps. 

Aneurism  needles,  sharp  and  blunt. 

Scalpels,        ) 

Keratomes,    >•  with  metal  handles. 

Trephines,     ) 

Surgical  needles. 

Needle  holder. 

Sterilise  these  before  use  by  boiling,  and  disinfect 
them  after  use  by  the  same  means.  Wipe  perfectly 
dry  immediately  the  disinfection  is  completed. 

4.  Anaesthetic. 

(a)  General:  The  safest  general  anaesthetic  for  animals 

is  an  A.  C.  E.  mixture,  containing  by  volume 
alcohol  i  part,  chloroform  2  parts,  ether  6  parts, 
and  should  be  administered  on  a  "cone"  formed 
by  twisting  up  one  corner  of  a  towel  and  placing 
a  wad  of  cotton-wool  inside  it. 

(b)  Local: 

Cocaine  hydrochloride,  2  per  cent,  solution. 
Eucaine,  2  per  cent,  solution. 

5.  Sterile  capsules  of  various  sizes. 

10  c.c.  (in  tenths  of  a  cu- 


centimetre). 

7.  Flasks  (75  c.c.)  containing  sterilised  normal  saline  solu- 

tion (or  sterile  bouillon). 

8.  Sterilised  cotton-  wool.     Cotton-  wool  is  packed  loosely 

in  a  copper  cylinder  similar  to  that  used  for  storing 
capsules,  and  sterilised  in  the  hot-air  oven. 

9.  Sterilised  gauze.     Gauze  is  sterilised  in  the  same  way 

as  cotton-wool. 

10.  Sterilised  silk  and  catgut  for  sutures.  These  are 
sterilised,  as  required,  by  boiling  for  some  ten 
minutes  in  the  water  steriliser. 


THE  PREPARATION  OF   THE   INOCULUM.  267 

11.  Flexible  collodion  (or  compound  tincture  of  benzoin). 

12.  Grease  pencil. 

13.  Tie-on  celluloid  labels,  to  affix  to  the  cages. 

14.  Razor. 

15.  Small  pot  of  warm  water. 

1 6.  Liquid   soap.     Liquid  soap  is  prepared   as  follows: 

Measure  out  100  grammes  of  soft  soap  and  add  to 
500  c.c.  of  2  per  cent,  lysol  solution  in  a  large  glass 
beaker;  dissolve  by  heating  in  a  water-bath  at 
about  90°  C.  Bottle  and  label  " Liquid  Soap." 

Material  Utilised  for  Inoculation. — The  material  in- 
oculated may  be  either— 

1 .  Cultures  of  bacteria — grown  in  fluid  media  or  on 
solid  media. 

2.  Metabolic  products  of  bacterial  activity — e.  g., 
toxins  in  solution. 

3.  Pathological  products  (fluid  secretions  and  excre- 
tions, solid  tissues). 

The  Preparation  of  the  Inoculum.— 
(a)   Cultivations  in  Fluid  Media.— 

1.  Flame  the  plug  of  the  culture  tube. 

2.  Remove  the  plug  and  flame  the  mouth  of  the 
tube. 

3.  Slightly  raise  the  lid  of  a  sterile  capsule,  insert 
the  mouth  of  the  culture  tube  into  the  aperture  and 
pour  some  of  the  cultivation  into  the  capsule. 

4.  Remove  the  mouth  of  the  culture  tube  from  the 
capsule,  replace  the  lid  of  the  latter,  flame  the  mouth 
of  the  tube,  and  replug. 

5.  Remove  the  syringe  from  the  steriliser,  squirt  out 
the  water  from  its  interior,  and  allow  to  cool. 

6.  Raise  the  lid  of  the  capsule  sufficiently  to  admit 
the   needle    of    the    syringe    and    draw  the  required 
amount  of  the  cultivation  into  the  barrel  of  the  syringe. 

(Or,  remove  a  definite  measured  quantity  of  the  cul- 
tivation directly  from  the  tube  or  flask  by  means  of 
a  sterile  graduated  pipette,  discharge  the  measured 
amount  into  a  sterile  capsule,  and  fill  into  the  syringe.) 


268      EXPERIMENTAL,  INOCULATION  OF  ANIMALS. 

If  it  is  necessary  to  introduce  a  large  bulk  of  fluid 
into  the  animal,  the  cultivation  should  be  transferred 
with  aseptic  precautions,  to  a  sterile  separatory  funnel, 
preferably  of  the  shape  shown  in  figure  141,  and  gradu- 
ated if  necessary.  This  is  supported  on  a  retort  stand 
and  raised  sufficiently  above  the  level  of  the  animal 
to  be  injected,  so  as  to  secure  a  good  "fall."  A  long 
piece  of  sterile  rubber  tubing,  fitted  with  an  injection 


Fig.  141. — Conical  separatory  funnel,  fitted  for  injection  of  fluid  cultivations. 

needle  and  provided  with  a  screw  clamp,  is  now  at- 
tached to  the  nozzle  of  the  funnel  and  the  operation 
completed  according  to  the  requirements  of  the  par- 
ticular case. 

If  the  injection  has  to  be  made  into  the  subcutaneous 
tissue  the  "fall"  may  not  be  sufficient  to  force  the 
fluid  in.  In  this  case  it  will  be  necessary  to  transfer 
the  culture  to  a  sterile  wash-bottle  and  fasten  a  rubber 


THE   PREPARATION    OF   THE    INOCULUM.  269 

hand  bellows  to  the  air  inlet  tube  (interposing  an 
air  filter)  and  attach  the  tubing  with  the  injection 
needle  to  the  outlet  tube  (Fig.  142).  By  careful  use 
sufficient  force  can  be  obtained  to  drive  the  injec- 
tion in. 

(6)  Cultivations  on  Solid  Media  (e.  g.,  Oblique  A  gar}. — 

1 .  By  means  of  a  sterile  graduated  pipette  introduce 
a  suitable  small  quantity  of  sterile  bouillon  (or  sterile 
normal  saline  solution)  into  the  culture  tube. 

2.  With  a  sterile  platinum  loop  or  spatula  scrape 
the  bacterial  growth  off  the  surface  of  the  medium, 


Fig.  142. — Arrangement  of  pressure  injection  apparatus. 

and  emulsify  it  with  the  bouillon.  It  then  becomes 
to  all  intents  and  purposes  a  fluid  inoculum. 

3.  Pour  the  emulsion  into  a  sterile  capsule  and  fill 
the  syringe  therefrom. 

Minimal  Lethal  Dose :  If  the  purpose  of  the  inocula- 
tion is  to  determine  the  minimal  lethal  dose,  a  slightly 
different  procedure  is  followed.  For  this  purpose  a 
special  platinum  loop  must  be  employed,  some  2.5  mm. 
by  0.75  mm.,  manufactured  with  parallel  sides,  and 
calibrated  by  careful  weighing.  (One  can  determine 
approximately  by  this  method  the  amount  of  bacterial 
growth  the  loop  will  hold  when  filled.) 


2JO      EXPERIMENTAL  INOCULATION  OF  ANIMALS. 

1.  The  cultivation    must  be    prepared  on  a  solid 
medium  of  the  optimum  reaction,  incubated  at  the 
optimum  temperature,  and  injected  at  the  period  of 
greatest  activity  and  vigour,  of  the  particular  organism 
it  is  desired  to  test. 

2.  Arrange  four  sterile  capsules  in  a  row  and  label 
them  I,  II,  III,  and  IV.     Into  the  first  deliver  10  c.c. 
sterile  bouillon  by  means  of  a  sterile  graduated  pipette ; 
and  into  each  of  the  remaining  three,  9.9  c.c. 

3.  Remove  one  loopful  of  the  bacterial  growth  from 
the  surface  of  the  medium  in  the  culture  tube,  observ- 
ing the  usual  precautions  against  contamination,  and 
emulsify  it  evenly  with  the  bouillon  in  the  first  capsule. 
Each  cubic  centimetre  of  the  emulsion  will  now  con- 
tain one-tenth  of    the    organisms    contained    in    the 
original  loopful  (written  shortly  o.  i  loop). 

4.  Remove  o.i  c.c.  of  the  emulsion  in  the  first  cap- 
sule  by   means   of   a   sterile   graduated   pipette   and 
transfer  it  to  the  second  capsule  and  mix  thoroughly. 
Drop  the  infected  pipette  into  a  jar  of  lysol  solution. 
This  makes  up  the  bulk  of  the  fluid  in  the  second  cap- 
sule to  10  c.c.,  and  therefore  every  cubic  centimetre 
of  bouillon  in  capsule  II  contains  o.ooi  loop. 

5.  Similarly,  o.i  c.c.  of  the  mixture  is  transferred 
from  capsule  II  to  capsule  III  (i  c.c.  of  bouillon  in 
capsule    III    contains   o.ooooi    loop),  and    then  from 
capsule  III  to  capsule  IV  (i  c.c.  of  bouillon  in  capsule 
IV  contains  o.ooooooi  loop). 

6.  With  sterile  graduated  pipettes  remove  the  neces- 
sary quantity  of  bouillon  corresponding  to  the  various 
divisors  of  ten  of  the  loop  from  the  respective  capsules, 
and  transfer  each  "dose"  to  a  separate  sterile  capsule 
and  label;  and  to  such  doses  as  do  not  amount  to  i  c.c. 
in  bulk,  add  the  necessary  quantity  of  sterile  bouillon. 

7.  Multiples  of  the  loop  are  prepared  by  emulsifying 
i,  2,  5,  or  10  loops  each  with  i  c.c.  sterile  bouillon  in 
separate  sterile  capsules. 


THE   PREPARATION    OF   THE   INOCULUM.  271 

8.  Inoculate  a  series  of  animals  with  these  measured 
doses,  filling  the  syringe  first  from  that  capsule  con- 
taining the  smallest  dose,  then  from  the  capsule  con- 
taining the  next  smallest,  and  so  on.     If  care  is  taken, 
it  will  not  be  found  necessary  to  sterilise  the  syringe 
during  the  series  of  inoculations. 

9.  Plant  tubes  of  gelatine  or  agar,  liquefied  by  heat, 
from  each  of  the  higher  dilutions,  say  from  o.ooooooi 
loop  to  o.oi  loop;  pour  plates  and  incubate.     When 
growth  is  visible  enumerate  the  number  of  organisms 
present  in  each,  average  up  and  calculate  the  number 
of  bacteria  present  in  one  loopful  of  the  inoculum. 

10.  The  smallest  dose  which  causes  the  infection  and 
death  of  the  animal  inoculated  is  noted  as  the  minimal 
lethal  dose  (written  shortly  m.  /.  d.). 

(c)  Toxins. — Prepared     by     previously     described 
methods  (vide  page  260),  are  manipulated  in  a  similar 
manner  to  cultivations  in  fluid  media. 

(d)  Pathological  Products. — Fluid  secretions,  excre- 
tions, etc.,  such  as  serous  exudation,  pus,  blood,  etc., 
are  collected  direct  from  the  body  in  sterile  capillary 
pipettes    (vide   Fig.    10,    page    21)    in    the    following 
manner: 

1.  Open  the  case  containing  the  pipettes,  grasp  one 
by  the  plugged  end,  remove  it  from  the  case,   and 
replace  the  lid  of  the  latter. 

2.  Pass  the  entire  length  of  the  pipette  twice  or 
thrice  through  the  flame  of  the  Bunsen  burner. 

3.  Snap  off  the  sealed  end  of  the  pipette  with  a  pair 
of  sterile  forceps. 

4.  Thrust  the  point  of  the  pipette  into  the  secretion, 
apply  the  mouth   to   the  plugged  end,   and  fill  the 
pipette  by  suction. 

5.  Seal  the  point  of  the  pipette  in  the  flame.     (If 
using  a  pipette  with  a  constriction  below  the  plugged 
mouthpiece,  this  portion  of  the  pipette  may  also  be 
sealed  in  the  flame.) 


EXPERIMENTAL  INOCULATION  OF  ANIMALS. 

When  about  to  perform  the  inoculation,  snap  the 
resealed  end  of  the  pipette  off  with  sterile  forceps  and 
blow  out  the  contents  of  the  pipette  into  a  sterile 
capsule,  from  which  the  injecting  syringe  is  filled. 

If  the  material  when  discharged  into  the  capsule  is 
very  thick  or  viscous,  a  small  quantity  of  sterile 
bouillon  or  normal  saline  solution  may  be  used  to 
dilute  it,  and  thorough  incorporation  effected  by  the 
help  of  a  sterile  platinum  rod. 

Solid  tissues,  such  as  spleen,  lymph  glands,  etc., 
may  be  divided  into  small  pieces  by  sterile  instruments- 
and  rubbed  up  in  a  sterilised  mortar  with  a  small 
quantity  of  sterile  bouillon  and  the  syringe  filled  from 
the  resulting  emulsion. 


Fig.  143. — Holding  rabbits  for  shaving. 

If  it  is  desired  to  inoculate  tissue  en  masse,  remove 
from  the  material  a  small  cube  of  i  or  2  mm.  and 
introduce  it  into  a  wound  made  by  sterile  instruments 
in  a  suitable  situation,  and  occlude  the  wounds  by 
means  of  a  sealed  dressing. 

Method  of  Securing  Animals  During  Inoculation.— 
For  the  majority  of  inoculations,  especially  when  no 
anaesthetic  is  administered,  it  is  customary  to  employ 
an  assistant  to  hold  the  animal  (see  Fig.  143);  but 
when  the  animal  is  anaesthetised,  it  is  more  convenient 
to  secure  it  firmly  to  some  simple  form  of  operating 
table,  such  as  Tatin's  (Fig.  144),  which  will  accommo- 
date rabbits,  guinea-pigs,  and  rats. 

A  useful  piece  of  apparatus,  too,  is  Voge's  holder 
for  guinea-pigs,  the  method  of  using  which  is  readily 
seen  from  the  accompanying  figure  (Fig.  145). 


SECURING   ANIMALS   DURING   INOCULATION.       273 


The  instrument  itself  consists  of  a  hollow  copper 
cylinder,  one  end  of  which  is  turned  over  a  ring  of  stout 
copper  wire,  and  from  this  open  end  a  slot  is  cut 


Fig.  144. — Operation  table. 

extending  about  half  way  along  one  side  of  the  cylinder. 
The  opposite  end  is  closed  by  a  "pull-off"  cap  and  is 
perforated  around  its  edge  by  a  row  of  ventilating 
holes,  which  correspond  with  holes  cut  in  the  rim  of 

the  cap.  In  the  event  of  the 
animal  resisting  attempts  to  re- 
move it  from  the  holder,  back- 
wards, this  cap  is  taken  off  and 
the  holder  placed  on  the  table 
and  the  guinea-pig  allowed  to 
walk  out. 


Fig.  145. — Taking  guinea- 
pig's  temperature. 


Fig.  146. — Voge' s  holder. 


To  provide  for  different-sized  animals,  two  sizes  of 
this  holder  will  be  found  useful : 

i.  Length,  1 6  cm.;  breadth,  6  cm.;  size  of  slot, 
8  cm.  by  2.5  cm. 


18 


274    EXPERIMENTAL  INOCULATION  OF  ANIMALS. 

2.  Length,  20  cm.;  breadth,  8  cm.;  size  of  slot, 
10  cm.  by  2.5  cm. 

A  convenient  holder  for  mice  and  even  small  rats 
is  shown  in  figure  147,  the  tail  being  securely  held  by 


Fig.  147. — Mouse  holder. 

the  spring  clip.  Needless  to  say,  the  holder  should 
be  entirely  of  metal,  and  the  wire  cage  detachable  and 
easily  renewed. 

METHODS  OF  INOCULATION.1 

The  following  methods  of  inoculation  apply  more 
particularly  to  the  rabbit,  but  from  them  it  will  readily 
be  seen  what  modifications  in  technique,  if  any,  are 
necessary  in  the  case  of  the  other  experimental  animals. 

1.  Cutaneous  Inoculation. — (Anaesthetic,  none.) 

1.  Have  the  animal  firmly  held  by  an  assistant  (or 
secured  to  the  operating  table). 

2.  Apply  the  liquid  soap  to  the  fur,  over  the  area 
selected  for  inoculation,  with   a  wad  of  cotton-wool, 
and  lather  freely  by  the  aid  of  warm  water;  shave 
carefully  and  thoroughly. 

3.  Wash  the  shaved  portion  of  skin  thoroughly  with 
2  per  cent,  lysol  solution. 

4.  Wash  off  the  lysol  with  rectified  spirit  and  allow 
the  alcohol  to  evaporate. 

1  In  the  United  Kingdom  a  special  licence  must  be  obtained  from  the  Sec- 
retary of  State  for  the  Home  Department,  granting  permission  to  its  holder  to 
perform  inoculation  upon  the  lower  animals. 


SUBCUTANEOUS   INOCULATION.  275 

5.  Make  numerous  short,  parallel,  superficial  inci- 
sions with  the  point  of  a  sterile  scalpel. 

6.  When  the  oozing  from  the  incisions  has  ceased, 
rub  the  inoculum  into  the  scarifications  by  means  of 
the  flat  of  a  scalpel  blade,  or  a  sterile  platinum  spatula. 

7.  Cover  the  inoculated  area  with  a  pad  of  sterile 
gauze  secured  in  situ  by  strips  of  adhesive  plaster  or 
by  sealing  down  the  edges  of  the  gauze  with  collodion. 

8.  Release  the  animal,  place  it  in  its  cage,  and  affix 
a  label  upon  which  is  written: 

(a)  Distinctive  name  or  number  of  the  animal. 

(b)  Its  weight. 

(c)  Particulars  as  to  source  and  dose  of  inoculum. 

(d)  Date  of  inoculation. 

2.  Subcutaneous  Inoculation. — 

(a)  Fluid  Inoculum. — (Anesthetic,  none.) 
Steps   1-4.  As  for  cutaneous  inoculation. 

5.  Pinch  up  a  fold  of  skin  between  the  forefinger 
and  thumb  of  the  left  hand;  take  the  charged  hypo- 
dermic syringe  in  the  left  hand,  enter  the  needle  into 
the  ridge  of  skin  between  the  finger  and  thumb,  and 
push  it  steadily  onwards  until  about  2  cm.  of  the  needle 
are  lying  in  the  subcutaneous  tissue.     Now  release 
the  grasp  of  the  left  hand  and  slowly  inject  the  fluid 
contained  in  the  syringe. 

6.  Withdraw  the  needle,  and  at  the  same  moment 
close  the  puncture  with  the  left  forefinger,  to  prevent 
the   escape   of   any   of   the   inoculum.     The   infected 
fluid,  unless  large  in  amount,  will  be  absorbed  within 
a  very  short  time. 

7.  Label,  etc. 

(b)  Solid  Inoculum. — (Anesthetic,  none.) 
Steps  1-4.  As  for  cutaneous  inoculation. 

5.  Raise  a  small  fold  of  skin  in  a  pair  of  forceps, 
and  make  a  small  incision  through  the  skin  with  a 
pair  of  sharp-pointed  scissors. 

6.  Insert  a  probe  through  the  opening  and  push  it 


276      EXPERIMENTAL,  INOCULATION  OF  ANIMALS. 

steadily  onwards  in  the  subcutaneous  tissue,  and  by 
lateral  movements  separate  the  skin  from  the  under- 
lying muscles  to  form  a  funnel-shaped  pocket  with  its 
apex  towards  the  point  of  entrance. 

7.  By  means  of  a  pair  of  fine-pointed  forceps  intro- 
duce a  small  piece  of  the  inoculum  into  this  pocket 
and  deposit  it  as  far  as  possible  from  the  point  of 
entrance. 

Or,  improvise  a  syringe  by  sliding  a  piece  of  glass 
rod  (to  serve  as  a  piston)  into  the  lumen  of  a  slightly 
shorter  length  of  glass  tubing  and  secure  in  position  by 
a  band  of  rubber  tubing.  Sterilise  by  boiling.  With- 
draw the  rod  a  few  millimetres  and  deposit  the  piece  of 
tissue  within  the  orifice  of  the  tube,  by  means  of  sterile 
forceps.  Now  pass  the  tube  into  the  depths  of  the 
''pocket,"  push  on  the  glass  rod  till  it  projects  beyond 


Fig.  148. — Glass  tube  syringe  for  subcutaneous  "  solid"  inoculation. 

the  end  of  the  tube,  and  withdraw  the  apparatus,  leav- 
ing the  tissue  behind  in  the  wound. 

8.  Close  the  wound  in  the  skin  with  a  dressing  of 
gauze  sealed  with  collodion  (or  tinct.  benzoin),  having 
previously  inserted  sutures,  if  necessary. 

9.  Label,  etc. 

3.  Intramuscular. — 

(a)  Fluid  Inoculum. — (Anesthetic,  none.) 

Steps  1-4.  As  for  cutaneous  inoculation. 

5.  Steady   the   skin   over   the   selected   muscle   or 
muscles   with   the   slightly   separated   left   forefinger 
and  thumb. 

6.  Thrust  the  needle  of  the  injecting  syringe  boldly 
into   the   muscular   tissue   and   inject   the   inoculum 
slowly. 

7.  Label,  etc. 


INTRAPERITONEAI,.  277 

(b)  Solid  Inoculum. — (Anesthetic,  A.  C.  E.} 

1.  Secure  the  animal  to  the  operation  table  and 
anaesthetise. 

2.  Shave  and  disinfect  the  skin  at  the  seat  of  opera- 
tion. 

3.  Surround  the  field  of  operation  by  strips  of  gauze 
wrung  out  in  2  per  cent,  lysol  solution. 

4.  Incise  skin,  aponeurosis,  and  muscle  in  turn. 

5.  Deposit  the  inoculum  in  the  depths  of  the  in- 
cision. 

6.  Close  the  wound  in  the  muscle  with  buried  sutures 
and  the  cutaneous  wound  with  either  continuous  or 
interrupted  sutures. 


Fig.  149. — Intraperitoneal  inoculation — fluid. 

7.  Apply  a  sealed  dressing  of  gauze  and  collodion. 

8.  Remove  the  animal  from  the  operating  table. 

9.  Label,  etc. 

4.  Intraperitoneal. — 

(a)  Fluid  Inoculum. — (Anaesthetic,  none.) 

Steps   1-4.  As  for  cutaneous  inoculation.  Shave  a 

fairly  broad   transverse  area,   stretching  from  flank 

to  flank. 

5.  Place  the  left  forefinger  on  one  flank  and  the 
thumb  on  the  opposite,  and  pinch  up  the  entire  thick- 
ness of  the  abdominal  parietes  in  a  triangular  fold. 
Now,  by  slipping  the  peritoneal  surfaces  (which  are  in 


278      EXPERIMENT AI,  INOCULATION  OF  ANIMALS. 

apposition)    one   over   the   other,    ascertain   that   no 
coils  of  intestine  are  included  in  the  fold. 

6.  Take  the  syringe  in  the  right  hand  and  with  the 
needle  transfix  the  fold  near  its  base  (Fig.  149). 

7.  Now  release  the  fold,  but  hold  the  syringe  steady; 
as  the  parietes  flatten  out,  the  point  of  the  needle  is 
left  free  in  the  peritoneal  cavity. 

8.  Inject  the  fluid  from  the  syringe. 

9.  Label,  etc. 
Second  Method: 

Steps  1-4.  As  in  the  first  method. 

5.  Heat  platinum  searing  wire  (0.5  mm.  wire, 
twisted  to  the  shape  indicated  in  figure  151,  mounted 
in  an  aluminium  handle)  to  redness,  and  with  it  burn 


Fig.    150. — Section   of  ab-        Fig.  151. — Platinum  wire  for  burning  hole 
dominal    wall,    etc.,    showing  through  parietes. 

point  of  needle  lying  free  above 
the  coils  of  intestine. 


a  hole  through  the  skin  and  abdominal  muscle  down 
to,  but  not  through,  the  visceral  peritoneum. 

6.  Fix    a   blunt-ended    needle    on   to    the   charged 
syringe,  and  by  pressing  the  rounded  end  firmly  against 
the  peritoneum  it  can  easily  be  pushed  through  into  the 
peritoneal  cavity. 

7.  Inject  the  fluid  from  the  syringe. 

8.  Label,  etc. 

This  method  is  especially  useful  when  it  is  desired 
to  collect  samples  of  the  peritoneal  fluid  from  time  to 
time  during  the  period  of  observation,  as  fluid  can  be 
removed  from  the  peritoneal  cavity,  at  intervals, 
through  this  aperture  in  the  abdominal  parietes,  by 
means  of  a  sterile  capillary  pipette. 


INTRAPERITONEAL.  279 

(b)  Solid  Inoculum  (or  the  inoculation  of  gelatine 
capsules  x  containing  fluid  cultivations). — (Anesthetic, 
A.  C.  E.) 

1.  Anaesthetise   the   animal   and   secure   it   to    the 
operating  table. 

2.  Shave  a  large  area  of  the  abdominal  parietes. 

3.  Make  an  incision  through  the  skin  in  the  middle 
line  about  2  cm.  in  length,  midway  between  the  lower 
end  of  the  sternum  and  the  pubes. 

4.  Divide  the  aponeuroses  between  the  recti  upon  a 
director. 

5.  Divide  the  peritoneum  upon  a  director. 

6.  Introduce    the    inoculum    into    the    peritoneal 
cavity. 

7.  Close   the  peritoneal  cavity  with  I,embert's  su- 
tures. 

8.  Close  the  skin  and  aponeurosis  incisions  together 
with  interrupted  sutures,  and  apply  a  sealed  dressing. 

9.  Release  the  animal  from  the  operating  table. 

10.  Label,  etc. 

1  Collodion  sacs  may  be  readily  prepared  by  the  following  method  : 

1.  Dip  a  small  test-tube  (5  by  0.5  cm.),  bottom  downwards,  into  a  beaker 
of  collodion,  and  dry  in  the  air  ;  repeat  this  process  three  or  four  times. 

2.  Dip  the  tube,  with  its  coating  of  collodion,  alternately  into  a  beaker  of 
alcohol   and  one  of  water.     This  loosens  the  collodion  and  allows  it  to  be 
peeled  off  in  the  shape  of  a  small  test-tube. 

3.  Take  a  20  cm.  length  of  glass  tubing,  of  about  the  diameter  of  the  test- 
tube  used  in  forming  the  sac,  and  insert  one  end  into  the  open  mouth  of  the  sac. 

4.  Suspend  the  glass  tube  with  attached  sac,  inside  a  larger  test-tube,  by 
packing  cotton-wool  in  the  mouth  of  the  test-tube  around  the  glass  tubing, 
and  place  in  the  incubator  at  37°  C.  for  twenty-four  hours.      When  removed 
from  the  incubator,  the  sac  will  be  firmly  adherent  to  the  extremity  of  the 
glass  tubing. 

5.  Plug  the  open  end  of  the  glass  tubing  with  cotton- wool,  and  sterilise  the 
test-tube  and  its  contents  in  the  hot-air  oven. 

To  use  the  sac,  remove  the  plug  from  the  glass  tubing,  partly  fill  the  sac 
with  cultivation  to  be  inoculated,  by  means  of  a  sterile  capillary  pipette,  and 
replug  the  tubing.  When  the  abdominal  cavity  has  been  opened,  remove 
the  tubing  and  attached  sac  from  the  protecting  test-tube,  close  the  sac  by 
tying  a  sterilised  silk  thread  tightly  around  it  a  little  below  the  end  of  the 
glass  tubing,  and  separate  it  from  the  tubing  by  cutting  through  the  collodion 
above  the  ligature,  and  the  sac  is  ready  for  insertion  in  the  peritoneal  cavity. 


280      EXPERIMENTAL  INOCULATION  OF  ANIMALS. 

5.  Intracranial. — (Anesthetic,  A.  C.  E.) 

1.  Anaesthetise   the    animal   and   secure   it   to   the 
operating  table,  dorsum  uppermost. 

2.  Shave  a  portion  of  the  scalp  immediately  in  front 
of  the  ears. 

3.  Mark  out  a  crescentic  flap  of  skin  muscle,  etc., 
convexity  forwards,  commencing  0.5  cm.  in  front  of 
the  root  of  one  ear  and  terminating  at  a  similar  spot 

in  front  of  the  other  ear.     Reflect  the 
marked  flap. 

4.  With  a  small  trephine  (diameter 
0.5  cm.)  remove  a  circular  piece  of  bone 
from  the  parietal  segment.  The  centre 
of  the  trephine  hole  should  be  at  the 
intersection  of  the  median  line  and  a 
line  joining  the  posterior  canthi  (Fig. 


5.  Introduce  the  inoculum  by  means 
of   a   hypodermic   syringe,   perforating 
the  dura  mater  with  the  needle  and  de- 
positing the  material  immediately  below 
this  membrane. 

6.  Turn  back  the  flap  of   skin  and 
r?ranil^ra"bbitra      secure  it  in  position  with  interrupted 

sutures. 

7.  Dress  with  sterile  gauze  and  wool  and  seal  the 
dressing  with  collodion. 
6.  Intraocular.— 
(a)  Fluid  Inoculum. — (Anesthetic,  cocaine.) 

1.  Instil  a  few  drops  of  a  sterile  solution  of  cocaine, 
2  per  cent,  (or  B-eucaine,  2  per  cent.),  and  repeat  the 
instillation  in  two  minutes. 

2.  Five  minutes  later  have  the  animal  firmly  held 
by  an  assistant. 

3.  Steady  the  eye  with  fixation  forceps;  then  pierce 
the  cornea  with  the  needle  of  the  syringe  and  make 
the  injection  into  the  anterior  chamber. 


INTRAVENOUS.  2  8 1 

4.  Label,  etc. 

(b)  Solid  Inoculum. — (Anesthetic,  A.  C.  E.) 

1.  Anaesthetise  the  animal  and  secure  it  firmly  to 
the  operating  table. 

2.  Irrigate   the    conjunctival   sac   thoroughly   with 
sterile  saline  solution. 

3.  Make  an  incision  through  the  upper  quadrant  of 
the  cornea  into  the  anterior  chamber  by  means  of  a 
triangular  keratome. 

4.  Seize  the  solid  inoculum  in  a  pair  of  iris  forceps, 
introduce  it  through  the  corneal  wound,  and  deposit 
it  on  the  anterior  surface  of  the  iris;  withdraw  the 
forceps. 

5.  Again  irrigate  the  sac  and  the  surface  of  the  cor- 
nea. 

6.  Release  the  animal  from  the  operating  table. 

7.  Label,  etc. 

7.  Intrapulmonary. — 

Fluid  Inoculum. — (Anaesthetic,  none.) 

1.  Have  the  animal  firmly  held  by  an  assistant. 
(In  this  case  the  foreleg  of  the  selected  side  is  drawn 
up  by  the  assistant  and  held  with  the  ear  of  that  side.) 

2.  Shave  carefully  in  the  axillary  line  and  disinfect 
the  denuded  skin. 

3.  Thrust  the  needle  of  the  syringe  boldly  through 
the  fifth  or  sixth  intercostal  space  into  the  lung  tissue. 

4.  Inject  the  contents  of  the  syringe  slowly. 

5.  Label,  etc. 

8.  Intravenous.— 

Fluid  Inoculum,. — (Anesthetic,  none.) 

Vein  selected,  posterior  auricular.  Although  this  is 
smaller  than  the  median  vein,  it  is  firmly  bound  down 
to  the  cartilage  of  the  ear  by  dense  connective  tissue, 
and  is  therefore  more  readily  accessible.  (In  the 
guinea-pig  the  jugular  vein  must  be  utilised,  and  in 
order  to  perform  the  inoculation  satisfactorily  a  gen- 
eral anaesthetic  must  be  administered  to  the  animal.) 


282   EXPERIMENTAL  INOCULATION  OF  ANIMALS. 

1.  Have  the  animal  firmly  held  by  an  assistant. 
The  selected  ear  is  grasped  at  its  root  and  stretched 
forwards  towards  the   operator. 

2.  Shave  the  posterior  border  of  the  dorsum  of  the 
ear. 

3.  Disinfect    the    skin    over    the    vein,    rubbing   it 
vigourously  with  cotton- wool  soaked  in  lysol.     The 
friction  will  make  the  vein  more  conspicuous.     Wash 
the  lysol  off  with  rectified  spirit  and  allow  the  latter 
to  evaporate. 

4.  Direct  the  assistant  to  compress  the  vein  at  the 
root  of  the  ear.     This  will  cause  its  peripheral  portion 
to  swell  up  and  increase  in  calibre. 


Fig.  153. — Intravenous  inoculation. 

Care  must  be  taken  in  preparing  the  inoculum,  as 
the  injection  of  even  small  fragments  may  cause  fatal 
embolism.  To  obviate  this  risk  the  fluid  should,  if 
possible,  be  filtered  through  sterile  filter  paper  before 
filling  into  the  syringe. 

Air  bubbles,  when  injected  into  a  vein,  frequently 
cause  immediate  death.  To  prevent  this,  the  syringe 
after  being  filled  should  be  held  in  the  vertical  posi- 
tion, needle  uppermost.  The  needle  should  be  thrust 
through  the  centre  of  a  piece  of  sterile  filter  paper  and 
the  piston  of  the  syringe  pressed  upwards  until  all  the 
air  is  expelled  from  the  barrel  and  needle.  Should  any 


INHALATION.  283 

drops  of  the  inoculum  be  forced  out,  they  will  fall 
on  the  filter  paper,  which  should  be  immediately 
burned. 

5.  Hold  the  syringe  as  one  would  a  pen  and  thrust 
the  point  of  the  needle  through  the  skin  and  the  wall 
of  the  vein  till  it  enters  the  lumen  of  the  vein  (Fig.  1 53) . 
Now  press  it  onwards  in  the  direction  of  the  blood 
stream — i.  e.t  towards  the  body  of  the  animal. 

6.  Direct   the   assistant   to   cease   compressing   the 
root  of  the  ear,  and  slowly  inject  the  inoculum.     (If 
the  fluid  is  being  forced  into  the  subcutaneous  tissue, 
a  condition  which  is  at  once  indicated  by  the  swelling 
that  occurs,  the  injection  must  be  stopped  and  another 
attempt  made.) 

7.  Withdraw  the  needle. 

8.  Label,  etc. 

9.  Inhalation. — 

(a)  Fluid  Inoculum. — (Anaesthetic,  none.) 

1.  Place  the  animal  in  a  closed  metal  box. 

2.  Through  a  hole  in  one  side  introduce  the  nozzle 
of  some  simple  spraying  apparatus,  such  as  is  used  for 
nasal  medicaments. 

3.  Fill  the  reservoir  of  the  instrument  (previously 
sterilised)   with  the  fluid  inoculum,   and  having  at- 
tached the  bellows,  spray  the  inoculum  into  the  interior 
of  the  box. 

4.  On  the  completion  of  the  spraying,  open  the  box, 
spray  the  animal  thoroughly  with  a  10  per  cent,  solution 
of  formaldehyde  (to  destroy  any  of  the  virus  that  may 
be  adhering  to  fur  or  feathers). 

5.  Transfer  the  animal  to  its  cage. 

6.  Label,  etc. 

7.  Thoroughly  disinfect  the  inhalation  chamber. 

(b)  Fluid  or  Powdered  Inoculum. — (Ancesthetic,  A.  C. 
E.) 

i.  Anaesthetise   the   animal  and  secure  it  firmly  to 
the  operating  table. 


284    EXPERIMENTAL  INOCULATION  OF  ANIMALS. 

2.  Pass  a  glass  tube  (previously  sterilised)  down  the 
larynx  into  the  trachea. 

3.  Connect  the  straight  portion  of  a  Y-shaped  piece 
of  tubing  to  the  upper  end  of  the  sterilised  tube  and 
couple  one  branch  of  the  Y  to  a  separatory  funnel  con- 
taining the  fluid  inoculum,  or  insufflator  containing  the 
powdered  inoculum,  and  the  other  to  a  hand  bellows. 

4.  Allow  the  fluid  inoculum  to  run  into  the  lungs  by 
gravity,  or  blow  in  the  powdered  inoculum  by  means 
of  a  rubber-ball  bellows. 

5.  Remove  the  intratracheal  tube;  release  the  animal 
from  the  table. 

6.  Label,  etc. 

As  an  alternative  method  in  the  case  of  fairly  large 
animals,  such  as  rabbits,  etc.,  a  sterile  piece  of  glass 
tubing  of  suitable  diameter  may  be  passed  through 
the  larynx  down  the  trachea  almost  to  its  bifurcation. 
Fluid  cultivations  may  then  be  literally  poured  into 
the  lungs,  or  cultivations,  dried  and  powdered,  may 
be  blown  into  the  lung  by  the  aid  of  a  small  hand 
bellows. 

One  other  method  of  inoculation  remains  to  be 
described,  which  does  not  require  operative  inter- 
ference. 

10.  Feeding.— 

1.  Fluid  Inoculum. — Small  pieces  of  sterilised  bread 
or  sop  (sterilised  in  the  steamer  at  100°  C.)  are  soaked 
in  the  fluid  inoculum  and  offered  to  the  animals  in  a 
sterile  Petri  dish  or  capsule. 

2.  Solid  Inoculum. — Small  pieces  of  tissue  are  placed 
in  sterile  vessels  and  offered  to  the  animals. 

Raising  the  Virulence  of   an  Organism.  —  If  it  is 

desired  to  raise  or  "  exalt"  the  virulence  of  a  feebly 
pathogenic  organism,  special  methods  of  inoculation 
are  necessary,  carefully  adjusted  to  the  exigencies  of 


ALTERING    THE   VIRULENCE.  285 

each   individual    case.     Among    the    most   important 
are  the  following: 

1 .  Passages  of  Virus. — The  inoculation  of  pure  culti- 
vations of  the  organism  into  highly  susceptible  animals, 
and  passing  it  as  rapidly  as  possible  from  animal  to 
animal,  always  selecting  that  method  of  inoculation — 
e.  q.,  intraperitoneal — which  places  the  organism  under 
the    most    favorable    conditions   for    its    growth    and 
multiplication. 

2.  Virus  Plus  Virulent  Organisms. — The  inoculation 
of  pure  cultivations  of  the  organism  together  with  pure 
cultivations   of   some   other   microbe   which   in   itself 
is  sufficiently  virulent  to  ensure  the  death  of  the  ex- 
perimental animal,  either  into  the  same  situation  or 
into  some  other  part  of  the  body.     By  this  association 
the  organism  of  low  virulence  will  frequently  acquire 
a  higher  degree  of  virulence,  which  may  be  still  further 
raised  by  means  of  "passages"  (vide  supra). 

3.  Virus    Plus    Toxins. — The    inoculation    of    pure 
cultivations  of  the  organism  into  some  selected  situa- 
tion, together  with  the  subcutaneous,  intraperitoneal, 
or  intravenous  injection  of  a  toxin, — e.  g.,  one  of  those 
elaborated  by  the  proteus  group, — either  simultane- 
ously with,  before,  or  immediately  after,  the  injection 
of  the  organism.     By  this  means  the  natural  resistance 
of  the  animal  is  lowered,  and  the  organism  inoculated 
is   enabled   to   multiply   and   produce   its   pathogenic 
effect,    its   virulence   being   subsequently   exalted   by 
means  of  "  passages." 

Attenuating  the  Virulence  of  an  Organism. — Attenu- 
ating or  lowering  the  virulence  of  a  pathogenic  microbe 
is  usually  attained  with  much  less  difficulty  than  the 
exaltation  of  its  virulence,  and  is  generally  effected 
by  influencing  the  environment  of  the  cultivations, 
as  for  example: 

i.  Cultivating  in  such  media  as  are  unsuitable  by 
reason  of  their  (a)  composition  or  (6)  reaction. 


286      EXPERIMENTAL,   INOCULATION   OF  ANIMALS. 

2.  Cultivating  in  suitable  media,  but  at  an  unsuitable 
temperature. 

3.  Cultivating  in  suitable  media,  but  in  an  unsuitable 
atmosphere. 

4.  Cultivation  in  suitable  media,  but  under  unfavor- 
able conditions  as  to  light,  motion,  etc. 

5.  By  a  combination  of  two  or  more  of  the  above 
methods. 


XVI.    POST-MORTEM  EXAMINATIONS 
OF  EXPERIMENTAL  ANIMALS. 

THE  post-mortem  examination  should  be  carried  out 
as  soon  as  possible  after  the  death  of  the  animal,  for  it 
must  be  remembered  that  even  in  cold  weather  the 
tissues  are  rapidly  invaded  by  numerous  bacteria 
derived  from  the  alimentary  tract  or  the  cavities  of 
the  body,  and  from  external  sources. 

The  following  outlines  refer  to  a  complete  and  ex- 
haustive necropsy,  and  in  routine  work  the  examina- 
tion will  rarely  need  to  be  carried  out  in  its  entirety. 
In  all  examinations,  however,  the  searing  irons  must 
be  freely  employed,  and  it  must  be  recollected  that  one 
instrument  is  only  to  be  employed  to  seize  or  cut  one 
structure.  This  done,  it  must  be  regarded  as  con- 
taminated and  a  fresh  instrument  taken  for  the  next 
step. 

Apparatus  Required : 

Steriliser  (vide  page  38). 

f  Scissors. 
Surgical  instruments:    -j  Forceps. 

(  Scalpels. 

Spear-headed  platinum  spatula  (Fig.  156). 
Searing  irons  (Fig.  154). 
Platinum  loop. 

Tubes  of  media — bouillon  and  oblique  agar. 
Grease  pencil. 
Sterile  capillary  pipettes. 
Sterile  capsules. 
Cover-slips. 

Bottles  of  fixing  fluid  (for  pieces  of  tissue  intended  for  sec- 
tioning) . 

i.  Place  the  various  instruments,  forceps,  scissors, 
scalpels,  etc.,  needed  for  the  autopsy  inside  the  steril- 
iser and  sterilise  by  boiling  for  ten  minutes;  then  raise 

287 


288 


POST-MORTEM    EXAMINATIONS. 


the  tray  from  the  steriliser,  close  the  lid  of  the  latter, 
and  rest  the  tray  on  it. 

2.  Heat  the  searing  irons  to  redness  in  a  separate  gas 
stove. 

3.  Fasten  the  body  of  the  animal,  ventral  surface 
upwards  (unless  there  is  some  special  reason  for  having 


Fig.  154- — Searing  iron. 

the  dorsum  exposed),  out  on  a  board  by  means  of 
copper  nails  driven  through  the  extremities. 

4.  Drench  the  fur  (or  feathers)  with  lysol  solution, 
2  per  cent.  This  serves  the  twofold  purpose  of  pre- 
venting the  hairs  from  flying  about  and  entering  the 
body  cavities  during  the  autopsy,  and  of  rendering 
innocuous  any  vermin  that  may  be  present  on  the 
animal. 


Fig.  155. — Apparatus  for  post-mortem  examination,  animal  on  board. 

5.  With  sterile  forceps  and  scalpel  incise  the  skin  in 
the  middle  line  from  the  top  of  the  sternum  to  the 
pubes.  Make  other  incisions  at  right  angles  to  the 
first  out  to  the  axillae  and  groins,  and  reflect  the  skin 
in  two  lateral  flaps.  (Place  the  now  infected  instru- 
ments on  the  board  by  the  side  of  the  body  or  support 
them  on  a  porcelain  knife  rest.) 


OF  EXPERIMENTAL  ANIMALS.         289 

6.  Inspect   the   seat   of  inoculation.     If  any   local 
lesion  is  visible,  sear  its  exposed  surface  and  remove 
material  to  make  cultivations  and  cover-slip  prepara- 
tions from  the  deeper  parts  by  means  of  the  platinum 
loop.     Collect  specimens  of  pus  or  other  exudation  in 
capillary  pipettes  for  subsequent  examination. 

7.  Sear  the  whole  of  the  exposed  surface  of  the 
thorax  with  the  searing  irons. 

8.  Divide  the  ribs  on  either  side  of  the  sternum  and 
remove  a  rectangular  portion  of  the  anterior  chest  wall 
with  sterile  scissors  and  a  fresh  pair  of  forceps,  expos- 
ing the  heart.     Place  the  infected  instruments  by  the 
side  of  the  first  set. 

9.  Raise  the  pericardial  sac  in  a  fresh  pair  of  forceps 
and  burn  through  this  structure  with  a  searing  iron. 

10.  Grasp  the  apex  of  the  heart  in  the  forceps  and 
sear  the  surface  of  the  right  ventricle. 

11.  Plunge  the  open  point  of  a  capillary  pipette 
through  the  seared  area  into  the  ventricle  and  fill 
with  blood. 

Make  cultivations  and  cover-slip  preparations  of  the 
heart  blood. 

12.  Sear  a  broad  track  in  the  middle  line  of  the  ab- 
dominal wall;  open  the  peritoneal  cavity  by  an  incision 
in  the  centre  of  the  seared  line. 

13.  Collect  a  specimen  of  the  peritoneal  fluid  (or  pus, 
if  present)  in  a  capillary  pipette.     Make  cultivations 
and  cover-slip  preparations  from  this  situation. 

14.  Collect  a  specimen  of  the  urine  from  the  dis- 
tended bladder  in  a  large  pipette  (in  the  manner  indi- 
cated for  heart  blood),  for  further  examination,  by 
cultivations,  microscopical  preparations,  and  chemical 
analysis. 

15.  Excise  the  spleen  and  place  it  in  a  sterile  capsule. 
(Sear  the  surface   of   this   organ;   plunge   the  spear- 
headed spatula  through  the  centre  of  the  seared  area, 
twist  it  round  between  the  finger  and  thumb,  and  re- 

19 


290  POST-MORTEM   EXAMINATIONS 

move  it  from  the  organ.  Sufficient  material  will  be 
brought  away  in  the  eye  in  its  head  to  make  cultiva- 
tions. A  repetition  of  the  process  will  afford  material 
for  cover-slip  preparations.) 

1 6.  In  like  manner  examine  the  other  organs — liver, 
lungs,  kidneys,  lymphatic  glands,  etc.     Prepare  culti- 
vations and  cover-slip  preparations. 

17.  Examine  the  other  cavities  of  the  body. 

1 8.  Remove  small  portions  of  various  organs  and 
place  in  separate  bottles  of  "fixing  fluid"  for  future 
sectioning.     Affix  to  each  bottle  a  label  bearing  all 
necessary  details  as  to  its  contents. 

19.  If  necessary,  remove  portions  of  the  organs  for 
preservation  and  display  as  museum  specimens  (vide 
page  292). 

20.  Gather  up  all  the  infected  instruments,  return 


Fig.  .156. — Spear-headed  spatula. 

them  to  the  steriliser,  and  disinfect  by  boiling  for  ten 
minutes. 

2 1 .  Cover  the  exposed  cavities  of  the  body  with  blot- 
ting or  filter  paper,  moistened  with  2  per  cent,  lysol 
solution. 

22.  Cremate  the  cadaver  together  with  the  board 
upon  which  it  is  fixed. 

23.  Stain   the   cover-slip   preparations   by   suitable 
methods  and  examine  microscopically. 

24.  Incubate  the  cultivations  and  examine  carefully 
from  day  to  day. 

25.  Make  full  notes  of  the  condition  of  the  various 
body   cavities    and   of   the   viscera   immediately   the 
autopsy  is  completed ;  and  add  the  result  of  the  micro- 
scopical and  cultural  investigation  when  available. 

26.  Finally,  the  results  of  the  action  of  the  organism 


OF    EXPERIMENTAL,    ANIMALS.  291 

or  organisms  isolated  may  be  summarised  under  the 
following  headings : 

Tissue  changes:1 

1.  Local — i.  e.,  produced  in  the  neighbourhood  of 

the  bacteria. 

Position:  (a)  At  primary  lesion. 
(b)  At  secondary  foci. 
Character :  (a)  Vascular  changes  and      .      , 

tissue  reactions. 

(b)  Degeneration       and  ch°ic 
necrosis. 

2.  General  (i.  e.,  produced  at  a  distance  from  the 

bacteria,  by  absorption  of  toxins) : 

(a)  In  special  tissues — e.  g.,  nerve  cells  and 

fibres,  secreting  cells,  vessel  walls,  etc. 

(b)  General  effects  of  malnutrition,  etc. 
Symptoms : 

(a)  Associated  with  known  tissue  changes. 

(b)  Without  known  tissue  changes. 

Permanent  Preparations — Museum  Specimens.— 

I.  Tube  Cultivations  of  Bacteria. — When  showing 
typical  appearances  these  may  be  preserved,  if  not 
permanently,  at  least  for  many  years,  as  museum 
specimens,  by  the  following  method: 

1.  Take  a  large  glass  jar  25  cm.  high  by  18  cm.  diam- 
eter, with  a  firm  base  and  a  broad  flange,  carefully 
ground,  around  the  mouth.     The  jar  must  be  fitted 
with  a  disc  of  plate  glass  ground  on  one  side,  to  serve 
as  a  lid. 

2.  Smear  a  layer  of  resin  ointment  (B.  P.)  on  the 
flange  around  the  mouth  of  the  jar. 

3.  Cover  the  bottom  of  the  jar  with  a  layer  of  cotton- 
wool and  saturate  it  with  formalin. 

4.  Remove  the  cotton- wool  plug  from  the  culture 
tubes  and  place  them,  mouth  upwards,  inside  the  jar. 
(If  water  of  condensation  is  present  in  any  of  the 
culture  tubes,  it  should  be  removed  by  means  of  a 

1  This  table  is  adapted  from  Muir  and  Ritchie's  "  Bacteriology." 


292 


POST-MORTEM    EXAMINATIONS 


capillary  pipette  before  placing  the  tubes  in  the  forma- 
lin chamber.) 

5.  Adjust  the  glass  disc,  ground  side  downwards, 
over  the  mouth  of  the  jar  and  secure  it  by  pressing  it 
firmly  down  into  the  ointment,  with  a  rotary  move- 
ment. 

6.  Remove  the  tubes  from  the  formalin  chamber 

after  the  lapse  of  a  week,  and  dry  the 
exterior  of  each. 

7.  Seal  the  open  mouth  of  each 
tube  in  the  blowpipe  flame  and  label. 

If  the  cultivations  are  intended  for 
museum  purposes  when  they  are  first 
planted,  it  is  more  convenient  to  em- 
ploy Bulloch's  tubes. 

These  are  slightly  longer  than  the 
ordinary  tubes,  and  are  provided 
with  a  constriction  some  2  cm.  below 
the  mouth  (Fig.  157) — a  feature 
which  renders  sealing  in  the  blow- 
pipe flame  an  easy  matter. 

//.  Tissues. — The  naked-eye  ap- 
pearances of  morbid  tissues  may  be 
preserved  by  the  following  method: 

1 .  Remove  the  tissue  or  organ  from 
the  cadaver,  using  great  care  to  avoid 
distortion  or  injury. 

2.  Place  it  in  a  wide-mouthed  stop- 
pered jar,  large  enough  to  hold  it  con- 
veniently, resting  on  a  pad  of  cotton- wool,  and  arrange 
it  in  the  position  it  is  intended  to  occupy  (but  if  it 
is  intended  to  show  a  section  of  the  tissue  or  organ, 
do  not  incise  it  yet). 

3.  Cover  with  the  Kaiserling  solution,  and  stopper 
the  jar. 

Kaiserling  solution  is  prepared  as  follows : 


Fig.  157. — Bulloch's 
tubes. 


OF   EXPERIMENTAL   ANIMALS.  293 

Weigh  out 

Potassium  acetate 30  grammes 

Potassium  nitrate 10        " 

and  dissolve  in 

Distilled  water 750  c.c. 

then  add 

Formalin 300    " 

Filter. 

4.  After   immersion    in    the    formalin    solution    for 
twenty-four  to  forty-eight  hours   (according  to  size), 
transfer  the  tissue  to  a  bath  of  methylated  spirit  for 
ten  minutes. 

5.  Remove  to  a  fresh  bath  of  spirit  and  watch  care- 
fully.    Immediately  the  natural  colours  show  in  their 
original  tints,  transfer  to  the  mounting  solution. 

The  mounting  solution  consists  of 

Glycerine , 500  c.c. 

Distilled  water 500    " 

Formalin I    " 

6.  After  twenty-four  hours  in  this  solution  transfer 
to  a  museum  jar,  fill  with  fresh  mounting  solution, 
and  seal. 

6a.  Or  transfer  to  museum  jar  and  fill  with  lique- 
fied gelatine,  to  which  has  been  added  i  per  cent, 
formalin.  Cover  the  jar  and  allow  the  gelatine  to  set. 
When  solid,  seal  the  jar  in  the  usual  manner. 


XVIL    OUTLINES  FOR  THE   STUDY  OF 
THE  PATHOGENIC  BACTERIA. 

(THE  outlines  here  given  for  the  study  of  the  patho- 
genic bacteria  are  those  in  use  in  the  author's  elemen- 
tary classes  for  medical  and  dental  students,  and  for 
those  qualifying  for  the  Public  Health  Service.  They 
represent  the  minimum  of  individual  practical  work 
necessary  for  the  acquisition  of  a  sound  knowledge 
of  the  more  important  specific  organisms  of  disease. 
At  the  same  time,  it  cannot  be  too  strongly  urged  that 
every  student  should,  if  possible,  work  out  each  organ- 
ism completely  and  thoroughly  on  the  lines  suggested 
in  the  Scheme  for  Study,  on  page  205.) 

The  student  who  has  conscientiously  worked  out 
the  methods,  etc.,  previously  dealt  with  is  in  a  position 
to  make  accurate  observations  and  to  write  precise 
descriptions  of  the  results  of  such  observations.  He 
is,  therefore,  now  entrusted  with  pure  cultivations  of 
the  various  pathogenic  bacteria,  in  order  that  he  may 
study  the  life-history  of  each  and  record  the  results 
of  his  own  observations — to  be  subsequently  corrected 
or  amplified  by  the  demonstrator.  In  this  way  he  is 
rendered  independent  of  text-book  descriptions,  the 
statements  in  which  he  is  otherwise  too  liable  to  take 
for  granted,  without  personally  attempting  to  verify 
their  accuracy.  For  this  reason  none  of  the  bacteria 
referred  to  in  the  following  pages  is  described  in  detail, 
nor  are  any  photomicrographic  reproductions  inserted. 

During  the  course  of  this  work  attention  is  also 
directed,  as  occasion  arises,  to  such  other  bacteria, 
pathogenic  or  saprophytic,  as  are  allied  to  the  par- 

294 


THE   ORGANISMS    OF   SUPPURATION.  295 

ticular  organisms  under  observation,  or  so  resemble 
them  as  to  be  possible  sources  of  error,  by  working 
them  through  on  parallel  lines.  By  this  means  a  fund 
of  information  is  obtained  with  regard  to  the  resem- 
blances and  differences,  morphological  and  cultural,  of 
a  large  number  of  bacteria. 

The  Organisms  of  Suppuration.— Whilst  nearly  all 
the  pathogenic  bacteria  possess  the  power,  under 
certain  conditions,  of  initiating  purely  pyogenic  pro- 
cesses in  place  of  or  in  addition  to  their  specific  lesions 
(e.  g.,  the  Bacillus  tuberculosis,  the  pneumococcus, 
etc.),  there  are  a  certain  few  organisms  which  commonly 
express  their  pathogenicity  in  the  formation  of  pus. 
These  are  usually  grouped  together  under  the  title 
of  "pyogenic  bacteria,"  as  distinct  from  those  which 
only  occasionally  exercise  a  pyogenic  role. 

The  organisms  included  under  this  heading  are: 

1.  Staphylococcus  pyogenes  albus. 

2.  Staphylococcus  pyogenes  aureus. 

3.  Staphylococcus  pyogenes  citreus. 

4.  Streptococcus  pyogenes  longus. 

5.  Micrococcus  tetragenus. 

6.  Micrococcus  gonorrhoeas. 

7.  Bacillus  pyocyaneus. 


296  OUTLINES   FOR   THE   STUDY   OF 

Staphylococcus      pyogenes     Micrococcus  candicans. 

albus.  Micrococcus  agilis. 

Staphylococcus     pyogenes 

aureus. 
Staphylococcus     pyogenes 

citreus. 

1.  Prepare  subcultivations  from  each: 

Bouillon,         ^ 

Agar  streak,    land  incubate  at  37°  C. 

Blood-serum, ) 

Agar  streak,  ' 

Gelatine  stab,        .,  .       ,  0  ~ 

and  incubate  at  20°  C. 
Potato, 

Litmus  milk,  t 

Compare  the  naked-eye  appearances  of  the  cultures 
from  day  to  day.  Note  M.  agilis  refuses  to  grow 
at  37°  C. 

2.  Make  hanging-drop  preparations  from  the  bouillon 
and  agar  cultivations  after  twenty-four  hours'  incuba- 
tion.    Examine  microscopically  and  compare. 

3.  Prepare  cover-slip  films  from  the  agar  cultures, 
after  twenty-four  hours'  incubation.     Stain  for  flagella 
by  the  modified  Pitfield's  method. 

4.  Make  microscopical  preparations  of  each  from  all 
the  various  media  after  twenty-four  and  forty-eight 
hours'    and    three    days'    incubation.     Stain    carbolic 
methylene-blue,    carbolic    fuchsin,    Gram's    method. 
Examine  microscopically  and  compare. 

5.  Stain  section  of  cardiac  muscle  provided  (staphy- 
lococcus  in  ulcerative  endocarditis)  by  Gram's  method, 
and  counterstain  with  eosin. 

6.  Stain  film  preparation  of  pus  from  an  abscess 
(containing    Staphylococcus    pyogenes   aureus)    with 
carbolic  methylene-blue  and  also  by  Gram's  method, 
counterstained  with  eosin. 

7.  Inoculate   a   white   mouse   subcutaneously  with 
three  loopfuls  of  a  forty-eight-hour  agar  cultivation 


THE    PATHOGENIC    BACTERIA.  297 

of  the  Staphylococcus  aureus,  emulsified  with  0.2  c.c. 
sterile  broth. 

Observe  carefully  during  life,  and  if  death  occurs 
make  a  careful  post-mortem  examination  (page  287). 


Gonococcus.  Micrococcus  tetragenus. 

1.  Prepare  subcultivations  of  each: 

Bouillon,        V 

Agar,  )  and  incubate  at  37°  C. 

L,itmus  milk,/ 

Gelatine  streak,)       .,  .       ,  0  ~ 

5  \  and  incubate  at  20°  C. 
Potato,  j 

Observe  the  culture  tubes  from  day  to  day. 
Note   that   the   gonococcus   refuses   to   grow   upon 
these  media  either  at  20°  C.  or  at  37°  C. 

2.  Prepare  duplicate  sets  of  subcultivations  of  the 
gonococcus : 

'incubate  one  set 
Ascitic  (or  serum)  bouillon,  0  ~ 

at  37°  C.  and 
Serum  agar,  ,-  ,, 

the  other  at 
Human  blood  agar.  0  ~ 

Examine  the  culture  tubes  from  day  to  day. 

Note  that  the  gonococcus  refuses  to  grow  at  20°  C. 

3.  Prepare  cover-slip  films  of  the  Micrococcus  tetra- 
genus from  all  the  media  and  of  the  gonococcus  from 
those  media  upon  which   it  grows,  after   twenty-four 
hours'  and  three  days'  incubation. 

Stain  carbolic  methylene-blue,  Gram's  method. 

4.  Double  stain  the  section  of  mouse's  spleen  (Micro- 
coccus    tetragenus)    with    picrocarmine    and    Gram's 
method. 

5.  Stain  cover-slip  film  preparations  of  urethral  pus 
(containing  gonococci)  with  carbolic  methylene-blue, 
capsule  stain,  Gram's  method,  counterstained  eosin. 

6.  Prepare  three  human   serum  agar  plates    (vide 


298  OUTLINES   FOR   THE   STUDY   OF 

page  198)  in  series  from  the  specimen  of  urethral  pus 
and  incubate  at  37°  C.  for  thirty-six  hours. 

Endeavour  to  isolate  the  M.  gonorrhoese  by  subculti- 
vating  suspicious  colonies  in  serum  agar  streak  culture. 

7.  Inoculate  a  white  mouse  subcutaneously  with 
two  loopfuls  of  a  two-day  agar  cultivation  of  the 
Micrococcus  tetragenus. 

Observe  until  death  takes  place,  then  make  a  com- 
plete post-mortem  examination. 


Streptococcus  pyo genes  Ion-     Streptococcus  brems. 

gus. 
Streptococcus      of      bovine 

mastitis. 
Diplococcus  pneumonia. 

1.  Prepare  subcultivations  from  each: 

Agar  streak, 

Blood  agar  streak, 

Bouillon,  and  incubate  at  37°  C. 

Litmus  milk, 

Potato, 

Gelatine  streak,       1 

-T.   .  fand  incubate  at  20°  C. 

Potato,  j 

Compare  the  naked-eye  appearances  of  the  cultures 
from  day  to  day. 

Note  that  the  pneumococcus  refuses  to  grow  at  20°  C. 

2.  Prepare  cover-slip  films  from  each  culture  tube 
after  twenty-four  and  forty-eight  hours'  incubation. 

Stain  carbolic  methylene-blue,  MacConkey's  capsule 
stain,  Gram's  method,  and  compare. 

3.  Examine  agar  cultivations  at  five  and  fourteen 
days  by  means  of  stained  preparations,  for  involution 
forms,   so-called   arthrospores,   etc. 

4.  Test  the  agar  cultures  for  spores  after  fourteen 
days'  incubation.     Result  negative. 


THE   PATHOGENIC    BACTERIA.  299 

5.  Stain  film  preparations  from  the  specimen  of  pus 
(containing    streptococcus)    carbolic    methylene-blue, 
Gram's  method,  counterstained  eosin. 

6.  Double  stain  the  sections  of  erysipelatous  skin, 
with  picrocarmine  and  Gram's  method. 

7.  Make   cover-slip   films   from   the   rusty   sputum 
(from  a  case  of  pneumonia)  and  stain  capsule  stain, 
and  Gram  counterstained  eosin. 

8.  Stain  the  section  of  pneumonia  lung  by  the  Gram- 
Weigert  method,  and  counterstain  with  eosin. 

9.  Inoculate  a  rabbit  intraperitoneally  with  a  loopful 
of  the  blood  agar  cultivation  of  the  pneumococcus. 

Observe  until  death  occurs,  recording  the  rectal 
temperature  at  frequent  intervals;  then  make  a  com- 
plete post-mortem  examination. 

Observe  the  capsulated  diplococci  in  the  heart  blood. 

10.  Inoculate  a  white  mouse  subcutaneously  with 
a  loopful  of  the  blood  agar  cultivation  of  the  Strepto- 
coccus longus. 

Observe  during  life,  and  when  death  occurs,  make 
a  complete  post-mortem  examination. 


Bacillus  of  Friedldnder.         B.  of  rhino  scleroma. 
(Pneumobacillus. ) 
i.  Prepare  subcultivations  of  each: 

Bouillon, 

Agar  streak, 


Blood-serum  streak, 
Potato, 
Litmus  milk. 
Gelatine  streak, 
Gelatine  stab, 


Incubate  at  37°  C. 


Incubate  at  20°  C. 


Glucose  gelatine  shake. 
Compare  the  naked-eye  appearances  of  the  cultures 
from  day  to  day. 


300  OUTLINES  FOR   THE   STUDY   OF 

2.  Make  hanging-drop  preparations  from  the  agar 
cultures  after  twenty-four  hours'  incubation. 

Examine  microscopically,  and  compare. 

3.  Make   film  preparations   of   each,   from   all   the 
various  media  after  twenty-four  hours'  and  three  days' 
incubation. 

Stain  carbolic  methylene-blue,  carbolic  fuchsin, 
Gram's  method.  Examine  microscopically  and  com- 
pare. 

Note  the  pleomorphism  and  involution  forms. 

4.  Test   the   agar   cultures   for   spores   after   seven 
days'  incubation.     Result  negative. 

5.  Stain  the  sections  of  mouse's  kidney  (containing 
pneumobacilli)  with  carbolic  methylene-blue. 

6.  Stain  the  sections  of  mouse's  spleen  (containing 
pneumobacilli)  with  capsule  stain. 

7.  Inoculate  a  mouse  subcutaneously  with  a  couple 
of  loopfuls  of  a  forty-eight-hour-old  potato  cultivation, 
emulsified  with  0.2  c.c.  sterile  bouillon. 

Observe  during  life,  and  after  death  make  a  com- 
plete post-mortem  examination. 

Note  the  well-defined  capsules  around  the  bacilli  in 
the  heart  blood. 


B.  pyocyaneus.  B.  fluorescens  liquefaciens. 

B.  fluorescens  non-liquefa- 

dens. 

I.  Prepare  subcultivations  of  each: 
Bouillon, 

Agar  streak,  ^ 

*     ,  -         Incubate  at  37°  C. 

Blood-serum  streak, 


Litmus  milk. 
Agar  streak, 
Gelatine  stab, 
Potato. 


Incubate  at  20°  C. 


THE   PATHOGENIC    BACTERIA.  301 

Compare  the  naked-eye  appearances  of  the  cultures 
from  day  to  day. 

Note  the  difference  in  the  "optimum"  temperatures. 

2.  Make  hanging-drop  preparations  from  the  agar 
and  bouillon  cultures  after  twenty -four  hours'  incuba- 
tion. 

Examine  microscopically,  and  compare. 

3.  Prepare  cultivations  of  each : 

(a)  Glucose  formate  bouillon,  and  incubate  anae- 
robically  at  37°  C.  and  20°  C. 

Compare  the  culture  tubes  after  three  days'  incuba- 
tion 

(b)  Nitrate  bouillon,  and  incubate  at  37°  C. 
Compare  the  gas  production  from  day  to  day. 

(c)  Glycerine  agar,  and  incubate  at  37°  C.  and  20°  C. 
Compare  the  pigment  production  at  five,  ten,  and 

fourteen  days. 

4.  Endeavour  to  obtain  solutions  of  the  pigment, 
formed  upon  agar  and  blood-serum  in  water,  chloro- 
form, alcohol. 

5.  Prepare  cover-glass  films  from  the  agar  cultiva- 
tions at  eighteen  to  twenty-four  hours. 

Stain  by  the  modifield  Pitfield  method. 

6.  Make  film  preparations  of  each  from  the  various 
media  after  twenty-four  hours'  and  three  days'  incu- 
bation. 

Stain    carbolic    methylene-blue,    carbolic    fuchsin, 
Gram's  method,  and  compare. 

7.  Stain    the    sections    of    guinea-pig's    spleen    (B. 
pyocyaneus)  with  carbolic  methylene-blue. 

8.  Make   a  complete  post-mortem  examination  of 
the  body  of  the  guinea-pig  (septicaemia  resulting  from 
pyocyaneus  infection). 


302  OUTLINES   FOR   THE   STUDY  OF 

Vibrio  cholerce.  Vibrio  metschnikom. 

Vibrio  of  Finkler  and  Prior. 
Spirillum  rubrum. 

i.  Prepare  subcultivations  of  each: 
Bouillon, 
Blood-serum  streak, 


Incubate  at  37° 
C. 


Agar  streak, 

Peptone  water, 

Nitrate  broth  (i  per  cent.), 

Litmus  milk. 

Gelatine  stab, )  _  0  ^ 

[  Incubate  at  20°  C. 
Potato.  J 

Compare  the  naked-eye  appearances  of  the  cultures, 
from  day  to  day. 

2.  Make  hanging-drop  preparations  from  the  bouillon 
and  agar  cultures  after  eighteen  hours'  incubation. 

Examine  microscopically,  and  compare. 

3.  Prepare  cover-glass   films   from  the  agar  culti- 
vations at  eighteen  to  twenty-four  hours. 

Stain  by  the  modified  Pitfield  method  (for  flagella). 

4.  Prepare  cover-slip  films  from  all  the  cultivations 
after  twenty-four  and  forty-eight  hours'  and  five  days' 
incubation. 

Stain  carbolic  methylene-blue,  carbolic  fuchsin 
(diluted  with  water),  by  Gram's  method.  Examine 
microscopically,  and  compare. 

5.  Test  the  agar  cultivations  for  spores  after  four- 
teen days'  incubation  at  37°  C.     Result  negative. 

6.  Test    the    peptone    water    cultivation    for    indol 
and  nitrite  after  two,  three,  and  five  days'  incubation. 

7.  Test  the  nitrate  bouillon  for  nitrites  after  three 
days'  incubation. 

8.  Test  a  twenty -four-hour-old  bouillon  cultivation 
against  the  serum  of  a  guinea-pig  that  has  been  im- 
munised against  the  Vibrio  cholerae. 

9.  Make  a  careful  post-mortem  examination  of  the 
guinea-pig  (killed  by  intraperitoneal  injection  of  the 
V.  cholerae). 


THE   PATHOGENIC   BACTERIA.  303 

Bacillus  diphtheria.  Bacillus  of  Hoffmann. 

(Klebs-Loffler  bacillus.)       Bacillus  of  xerosis. 
i.  Prepare  subcultivations  of  each: 
Agar  streak, 
Blood-serum  streak, 


Potato, 


Incubate  at  37°  C. 


Bouillon, 
Litmus  milk. 
Gelatine  streak.     Incubate  at  20°  C. 
Compare  the  naked-eye  appearance  of  the  cultures 
from  day  to  day. 

2.  Make  hanging-drop  preparations  from  the  bouil- 
lon  and   agar   cultivations   after   twenty-four   hours' 
incubation. 

Examine  microscopically,  and  compare. 

3.  Make  cover-slip  films  from  the  blood-serum  cul- 
tures, after  twelve  to  eighteen  hours'  incubation. 

Stain  Neisser's  method. 

4.  Make  film  preparations  of  each  from  all  the  media 
after    twenty-four    and    forty-eight    hours'    and    five 
days'  incubation. 

Stain  carbolic  methylene-blue,  carbolic  fuchsin, 
Gram's  method,  Neisser's  method,  and  compare. 

5.  Test  the  agar  cultivations  for  spores,  after  four- 
teen days'  incubation.     Result  negative. 

6.  Stain  the  sections  of  diphtheritic  membrane  (a) 
with  carbolic  methylene-blue,  and  (6)  by  the  Gram- 
Weigert  method — to  demonstrate  the  fibrin. 

Note  the  diphtheria  bacilli  in  the  superficial  layers  of 
the  membrane,  often  arranged  in  clusters  and  masses. 

7.  Inoculate   a   guinea-pig   under   the   skin   of   the 
abdomen  with  i  c.c.  of  a  forty-eight-hour-old  bouillon 
cultivation  of  the  B.  diphtherias. 

Observe  carefully  until  death  occurs,  then  make 
post-mortem  examination. 

Prepare  cultivations  on  blood-serum  from  (a)  local 
lesion,  (6)  heart  blood,  and  incubate  at  37°  C. 


304  OUTLINES   FOR   THE   STUDY   OF 

Note  that  a  cultivation  from  a  yields  a  growth  of 
the  B.  diphtheriae,  while  that  from  b  does  not. 

Prepare  cover-glass  films  from  local  lesion.  Note 
the  bacilli  present. 


B.  typhi  abdominalis.  B.  coli  communis. 

B.  enter itidis  (of  Gaertner) . 
B.  aquatilis  sulcatus. 
i.  Prepare  subcultivations  of  each: 
Bouillon, 

Litmus  milk,  ^ 

Incubate  at  37°  C. 
Peptone  water, 


Agar  streak. 

Bouillon, 

Potato, 


Incubate  at  20°  C. 


Gelatine  streak. 
Compare  the  naked-eye  appearances  of  the  cultures 
from  day  to  day. 

Note  that  B.  aquatilis  sulcatus  will  not  grow  at  37°  C. 

2.  Make  hanging-drop  preparations  from  the  bouillon 
and  agar  cultures  after  eighteen  hours'  incubation. 

Examine  microscopically,  and  compare. 

3.  Make  cover-slip  preparations  from  the  agar  culti- 
vations  after  eighteen   hours'   incubation. 

Stain  modified  Pitfield's  method. 

4.  Make  cover-slip  preparations  of  each  from  all  the 
media  after  twenty-four  hours'  and  five  days'  incuba- 
tion. 

Stain    carbolic    methylene-blue,     Gram's    method, 
modified  Pitfield's  method,  and  compare. 

5.  Test  agar  cultivations  at  fourteen  days  for  spores. 
Result  negative. 

6.  Test  the  peptone  water  cultivations  at  five  days 
for  indol. 


THE   PATHOGENIC    BACTERIA.  305 

7.  Prepare  glucose-formate-gelatine  "shake"  culti- 
vations and  incubate  at  20°  C. 

Compare  from  day  to  day. 

8.  Pour  gelatine  plate  cultivations  of  each  and  in- 
cubate at  20°  C.,  for  three  or  four  days. 

Compare  the  colonies,  naked  eye  and  under  a  i-inch 
lens. 

9.  Stain  the  sections  of  typhoid  ulcer  with  carbolic 
methylene-blue. 

10.  Stain  sections   of  guinea-pig  spleen    (B.   typhi 
abdominalis)  with  dilute  carbolic  fuchsin. 

1 1 .  Prepare  a  bouillon  cultivation  of  each  and  incu- 
bate at  20°  C.  for  twenty-four  hours. 

Test    each    culture    against    blood-serum    from    a 
typhoid  patient. 


Bacillus  anthracis.  Bacillus  subtilis. 

Bacillus  mycoides. 

1.  Prepare  subcultivations   of  each:  • 

Bouillon, 

Agar  streak, 

T  ?  Incubate  at  37°  C. 

Litmus  milk, 

Blood-serum  streak. 

Gelatine  stab.) 

Potato.  '[  Incubate  at  20°  C. 

Compare  the  naked-eye  appearances  of  the  cultures 
from  day  to  day. 

2.  Make  hanging-drop  preparations  from  the  bouillon 
and  agar  cultivations  after  twenty-four  hours v  incuba- 
tion. 

3.  Make  cover-slip  preparations  from  the  agar  culti- 
vations after  twenty-four  hours'  incubation. 

Stain    modified    Pitfield,    examine    microscopically, 
and  compare. 

4.  Make  cover-slip  preparations  from  the  various 

20 


306  OUTLINES   FOR  THE   STUDY   OF 

media  after  twenty-four  and  forty-eight  hours'  and 
seven  days'  incubation. 

Stain  carbolic  methylene-blue,  carbolic  fuchsin, 
Gram's  method,  and  compare. 

5.  Prepare  cultivations  of  each,  in  nitrate  broth, 
incubate  three  days  at  37°  C.,  and  test  for  nitrites. 

6.  Prepare  cultivations  of  each,  in  peptone  water, 
incubate  three  days  at  37°  C.,  and  test  for  indol. 

7.  Test  the  agar  cultivations  for  spores,  after  seven 
days'  incubation. 

8.  Prepare  cover-glass  films  from  these  agar  cultiva- 
tions and  stain  for  spores  by  method  2  (vide  page    91). 

9.  Double  stain  the  sections  of  malignant  pustule 
(from  man,   containing  anthrax  bacilli)   with   picro- 
carmine  and  Gram's  method. 

10.  Stain  the  sections  of  guinea-pig's  lung  (contain- 
ing B.  anthracis)  with  carbolic  methylene-blue. 

IT.  Inoculate  a  guinea-pig  subcutaneously  with  two 
loopfuls  of  a  forty-eight-hour-old  agar  cultivation  of  the 
B.  anthracis,  emulsified  with  i  c.c.  sterile  bouillon. 

Observe  carefully  during  life. 

When  dead,  make  complete  post-mortem  examina- 
tion. 


Bacillus  tuberculosis.  Bacillus  of  aman  tubercle. 

Bacillus  phlei. 

(Timothy  grass  bacillus.) 

i.  Prepare  subcultivations  of  th'e  tubercle  bacillus 
and  that  of  avian  tubercle: 

Blood-serum  streak, 
*  Agar  streak, 

Glycerine  agar  streak,  _ 

^/  '    .„  Incubate  at  37°  C. 

Glycerine  bouillon, 

Litmus  milk, 
Glycerinated  potato. 

Glycerine  agar  streak, ) 

~J*\.  [ Incubate  at 

Gelatine  streak.  J 


THE  PATHOGENIC  BACTERIA. 


307 


Fig.   158. 


308  OUTLINES   FOR   THE   STUDY   OF 

Compare  the  culture  tubes  from  day  to  day. 
Note  that  no  growth  takes  place  at  20°  C. 

2.  Prepare  subcultivations  of  the  B.  phlei  upon  all 
the  ordinary  media,  and  incubate  at  37°  C.  and  20°  C. 

3.  Make  cover-slip  preparations  of  each,  from   all 
the  various  media  after  twenty-four  hours'  and  seven 
days'  incubation. 

Stain  carbolic  methylene-blue,  carbolic  fuchsin 
(films  of  each  organism  should  be  stained  in  the  usual 
manner,  and  also  by  immersion  in  the  stain  for  four 
hours),  Ziehl-Neelsen  method,  Gram's  method.  (Em- 
ploy heat  in  staining  with  the  aniline  gentian  violet, 
as  is  done  in  the  Ziehl-Neelsen  method,  also  warm  the 
iodine  solution.) 

4.  Double  stain  the  sections  of  guinea-pig's  spleen 
(B.  tuberculosis)  with  picrocarmine  and  Ziehl-Neelsen 
method. 

5.  Stain  the  sections  of  lung  (miliary  tuberculosis) 
by   the   Ziehl-Neelsen   method,    counter  staining   with 
Bismarck  brown. 

6.  Make  a  careful  post-mortem  examination  of  the 
guinea-pig  which  has  succumbed  to  general  tubercu- 
losis, as  the  result  of  subcutaneous  inoculation  at  the 
inner  aspect  of  the  bend  of  the  left  knee. 

NOTE. — Every  post-mortem  examination  of  animals 
infected  with  tuberculous  material  should  include  the 
naked-eye  and  microscopical  examination  of  the 
popliteal,  superficial,  and  deep  inguinal,  iliac,  lumbar, 
and  axillary  glands  on  each  side  of  the  body,  also  the 
retrohepatic,  bronchial,  and  sternal  glands,  the  spleen, 
liver,  and  lungs  (see  Fig.  158), 


Bacillus  lepra. 

i.  Stain  the  sections   of    (a)   ulnar  nerve  and    (6) 
leprous  nodule  by  the  Ziehl-Neelsen  method 

This  organism  cannot  yet  be  cultivated  artificially. 


THE   PATHOGENIC   BACTERIA.  309 

Bacillus  tetani. 

Bacillus  cedematis  maligni. 

Bacillus  of  symptomatic  anthrax. 

B.  enteritidis  sporogenes. 

B.  botulinus. 

1.  Prepare  agar  streak  subcultivations  from  each  and 
incubate  aerobically  at  37°  C. 

Observe  the  culture  tubes  until  the  completion  of 
seven  days*  incubation.  Result,  no  growth. 

2.  Prepare  bouillon  cultivations  of — 

Bacillus  tetani  and  immediately  inoculate  the  cul- 
ture tube  with  B.  prodigiosus. 

Bacillus  cedematis  maligni  and  immediately  inocu- 
late the  culture  tube  with  Staphy  loco  ecus  aureus. 

Bacillus  of  symptomatic  anthrax  and  immediately 
inoculate  the  culture  tube  with  B.  pyocyaneus. 

B.  enteritidis  sporogenes  and  immediately  inoculate 
the  culture  tube  with  B.  subtilis. 

Incubate  these  cultivations  aerobically  at  37°  C. 

Observe  the  symbiotic  growth  of  the  anaerobes. 

3.  Prepare  subcultivations  from  each: 

/Incubate  anae- 

Glucose  formate  agar  streak,  \     ro^lcaHy    at 

Blood-serum  streak.  )     £7  tl      t  fm 

/     Bulloch's 

\     apparatus). 

Incubate  anaero- 
Glucose  formate  bouillon,        «.     ,,  0 

bically  at   37° 

Glucose  formate  agar  stab,       ~      ,.       -r,     ; 
T.,  M1  C.    (in    Buch- 

Litmus  milk.  ,    L  i_     \ 

ner  s  tubes). 

Incubate    an- 
aerobically 


Glucose  formate  gelatine  stab. 


Buchner's 
tube). 

4.  Make  hanging-drop  preparations  in  the  ordinary 
way  and  examine  microscopically. 


at  37°  C.  (in 


310  OUTLINES   FOR  THE   STUDY   OF 

Make  another  set  of  hanging-drop  preparations,  but 
before  sealing  the  cell  introduce  a  few  crystals  of 
pyrogallic  acid  and  a  drop  of  caustic  soda  solution, 
to  absorb  the  oxygen  and  render  the  hanging  drop 
"anaerobic."  Examine  microscopically. 

5.  Prepare  cover-slip   films   from   the   agar   streak 
cultivations  at  twenty-four  hours. 

Stain  modified  Pitfield  (for  flagella). 

6.  Prepare  cover-slip  films  from  each  of  the  various 
media  after  forty-eight  hours'  and  three  days'  incu- 
bation. 

Stain  carbolic  methylene-blue  and  Gram's  method. 

7.  Prepare  cover-slip  films  from  the  agar  cultiva- 
tions after  seven  days'  incubation. 

Stain  by  Muller's  method  (for  spores). 

8.  Make  careful  post-mortem   examinations  of  the 
guinea-pigs  which  have  died  as  the  result  of  infection 
with   the   B.    tetani,  B.    maligni,    and   B.   enteritidis 
sporogenes,  respectively. 


Bacillus  pestis.  B.   septiccemia   h&morrha- 

gicce. 
B.  suipestifer. 

(Bacillus  of  hog  cholera.) 
i.  Prepare  subcultivations  from  each: 
Bouillon, 


Agar  streak, 


and  incubate  at  37°  C. 


Litmus  milk, 
Gelatine  streak, 
Gelatine  stab, 


and  incubate  at  20°  C. 


•Potato, 

Compare  the  naked-eye  characters  of  the  cultures 
from  day  to  day. 


THE  PATHOGENIC   BACTERIA.  311 

2.  Add  a  loopful  of  sterile  vaseline  to  a  tube  of 
nutrient  bouillon. 

Prepare  a  subcultivation  of  the  B.  pestis  in  this  and 
incubate  at  37°  C. 

Note  the  formation  of  stalactites  depending  from 
the  fat  globules. 

3.  Make  hanging-drop  preparations  from  the  bouillon 
and  agar  cultures,  after  forty-eight  hours'  incubation. 
Examine  microscopically  and  compare. 

4.  Prepare  cover-slip  film  preparations  of  each  from 
all   the   media   after  twenty-four,   forty-eight   hours' 
and  three  days'  incubation. 

Stain  carbolic  methylene-blue,  carbolic  thionine  blue, 
Gram's  method,  and  compare. 

5.  Prepare  smear  cultivations  on  the  surface  of  very 
dry  agar  and  incubate  at  37°  C.  for  forty-eight  hours. 

Make  cover-slip  preparations  from  the  growth  and 
stain  with  carbolic  thionine  blue. 

Note  the  involution  forms  of  B.  pestis. 

6.  Examine  the  agar  streak  cultivations,  after  seven 
days'  incubation,  for  spores. 

7.  Sterilise  some  salt,  in  a  glass  capsule,  in  the  hot- 
air  oven. 

Add  about  0.5  gramme  to  a  tube  of  nutrient  bouillon 
and  incubate  at  37°  C.  for  forty-eight  hours,  to  deter- 
mine its  sterility. 

8.  Inoculate  this  bouillon  with  the  B.  pestis  and 
incubate  at  37°  C.  for  twenty-four  hours. 

Examine  the  resulting  growth  microscopically. 
Note  the  plasmolysed  bacilli. 

9.  Stain  the  sections  of  bubo  (containing  B.  pestis) 
with    (a)    Loftier 's    methylene-blue    and  (b)  carbolic 
thionine  blue. 

10.  Make  a  complete  post-mortem  examination  of 
the  cadaver  of  the  rat  infected  intraperitoneally  with 
the  B.  pestis. 


312  OUTLINES   FOR   THE   STUDY   OF 

Streptothrix  actinomycotica.  Cladothrix  nivea. 

1.  Prepare  subcultivations  from  each: 

Bouillon,  \ 

Glycerine  bouillon,) 

Agar,  land  incubate  at  37°  C. 

Glycerine  agar,        \ 

Litmus  milk,  / 

Gelatine  streak,) 

p  Y  and  incubate  at  20°  C. 

Compare  the  naked-eye  appearances  of  the  cultures 
from  day  to  day. 

2.  Prepare  cover-slip  film  preparations  of  each  from 
all  the  media  after  twenty-four  hours'  and  three  days' 
incubation. 

Stain  carbolic  methylene-blue,  carbolic  fuchsin, 
Gram's  method.  Examine  microscopically  and  com- 
pare. 

3.  Double  stain  sections  of  human  liver   (affected 
with  actinomycosis)  with  picrocarmine  and  the  Gram- 
Weigert  method. 

4.  Stain  the  section  of  cow's  tongue  (infected  with 
actinomyces)    by    Gram's    method    and  counterstain 
with  dilute  aqueous  solution  of  fuchsin. 

5.  Examine  the  pus  from  a  breaking-down  nodule 
(from  cow's  tongue)  with  a  hand  lens,  pick  out  some 
of  the  minute  yellowish  granules  with  a  sterile  platinum 
loop,  and  transfer  to  a  sterile  capsule. 

6.  Crush  the  selected  granules  with  a  sterile  glass 
rod.     Make    cover-slip    preparations    and    stain    by 
Gram's  method. 

7.  Inoculate  four  tubes  of  glycerine  agar  in  series 
with  some  of  the  material. 

Observe  the  resulting  growth  macroscopically  and 
microscopically. 


[  and  incubate  at  20°  C. 


THE   PATHOGENIC   BACTERIA.  313 

B.  influenzas.  Bacillus  cegyptiacus. 

(Koch-Week's  bacillus.) 

1.  Prepare  subcultivations  of  each: 

Bouillon,  \ 

Serum  bouillon,) 

Agar,  }and  incubate  at  37    C. 

Blood  agar, 

Serum  agar, 
Observe  from  day  to  day. 

Note  that  growth  only  occurs  in  serum  and  blood 
media. 

2.  Prepare  subcultivations  from  each: 

Serum  bouillon  ^ 

Blood  agar, 
Observe  from  day  to  day  for  four  days. 
Note  that  no  growth  takes  place. 

3.  Transfer  the  subcultivations   (section  2)  to  the 
incubator  at  37°  C. 

Note  that  no  growth  takes  place — the  bacilli  are 
dead. 

4.  Prepare  cover-slip  films  of  each  bacillus  from  all 
the  media  upon  which  growth  takes  place,  after  six- 
teen, twenty-four,  and  forty-eight  hours'  incubation. 

Stain  carbolic  methylene-blue,  very  dilute  aqueous 
solution  of  fuchsin,  Gram's  method.  Examine  micro- 
scopically and  compare. 

5.  Prepare  cover-slip  preparations  of  the  yellowish, 
more  solid  masses  of  the  specimen  of  sputum  from  a 
case  of  influenza. 

Stain  in  dilute  fuchsin  solution  for  twenty  minutes. 
Examine  microscopically. 

6.  Wash  some  of  these  selected  portions  of  sputum 
in  several  changes  of  sterile  distilled  water. 

7.  Inoculate  four  blood  agar  tubes  in  series  from  the 
washed  sputum,  and  incubate  at  37°  C. 

Observe  the  resulting  growth  macroscopically  and 
microscopically. 


314  OUTLINES   FOR   THE   STUDY   OF 

8.  Prepare  cover-slip  films  of  pus  from  a  case  of 
acute  muco-purulent  conjunctivitis. 

Stain  carbolic  methylene-blue,  dilute  fuchsin  solution. 

9.  Inoculate  four  blood  agar  tubes  in  series  from  the 
pus  from  cases  of  acute  conjunctivitis  and  incubate 
at    37°    C.     Observe    the    resulting    growth     macro- 
scopically  and  microscopically. 


Bacillus  mallei. 

1.  Prepare  subcultivations : 

Bouillon, 

Agar  smear, 

Blood-serum,  and  lncubate  at  ^    C" 

Litmus  milk, 

Gelatine  streak,) 

-PJ  ,  \  and  incubate  at  20   C. 

Potato,  j 

Observe  the  naked-eye  appearances  of  the  cultures 
from  day  to  day. 

2.  Make  hanging-drop  preparations  from  the  bouillon 
and  agar  cultivations. 

3.  Prepare  cover-slip  films  from  the  agar  and  blood- 
serum  cultivations,  after  eighteen  hours'  and  three  days' 
incubation. 

Stain  carbolic  methylene-blue,  Neisser's  method, 
and  by  Gram's  method.  Examine  microscopically 
and  compare. 

Also  compare  with  corresponding  preparations  of 
the  B.  diphtheriae. 

4.  Stain  sections  of  the  testicle  of  the  guinea-pig 
(containing  B.  mallei),  by  over  staining  with  Nicolle's 
gentian  violet   (vide  page   83),  and   subsequently  de- 
colourising with  i  per  cent,  acetic  acid. 

5.  Stain   the   sections   of   horse's   lung    (containing 
glanders  bacilli)  with  LofBer's  methylene-blue. 


THE    PATHOGENIC    BACTERIA.  315 

6.  Make  a  complete  post-mortem  examination  of 
the  guinea-pig  killed  by  infecting  subcutaneously  with 
the  B.  mallei. 


Micrococcus  meiitensis. 
i.  Prepare  subcultivations : 

Bouillon,  \ 

Glycerine  agar  smear,) 

Brain  agar  smear,        >  Incubate  at  37°  C. 

Blood-serum,  \ 

Litmus  milk.  / 

Agar  streak, 


Gelatine  streak, 


Incubate  at  20°  C. 


Potato. 

Observe  the  naked-eye  appearances  of  the  cultures 
from  day  to  day. 

Note  the  extremely  scanty  growth  on  gelatine  and 
agar  at  20°  C. 

2.  Make  hanging-drop  preparations  from  the  bouillon 
and  agar  cultivations. 

3.  Prepare  cover-slip  films  from  the  growth  on  each 
of    the    media    and    stain    carbolic    methylene-blue, 
dilute  solution  of  fuchsin,  by  Gram's  method;  examine 
microscopically  and  compare. 

Note  the  bacillary  forms  on  agar. 

4.  Examine  the  agar  cultivations,  after  seven  days' 
incubation,  for  spores.     Result  negative. 

5.  Test    a    forty-eight-hour-old    bouillon     culture, 
filtered  through  a  sterile  filter  paper,  against  the  serum 
of  a  patient  suffering  from  Malta  fever. 


XVHL  BACTERIOLOGICAL  ANALYSES. 

EACH  bacteriological  or  bacterioscopical  analysis  of 
air,  earth,  sewage,  various  food-stuffs,  etc.,  includes, 
as  a  general  rule,  two  distinct  investigations: 

1.  Quantitative. 

2.  Qualitative. 

The  first  aims  simply  at  enumerating  (approximately) 
the  total  number  of  bacteria  present  in  any  given  unit 
of  volume  irrespective  of  the  nature  and  character  of 
individual  organisms. 

The  second  seeks  to  classify  the  bacteria  found, 
and  to  accurately  identify  individual  organisms.  As  a 
subdivision  of  the  qualitative  examination,  an  esti- 
mation is  often  made,  and  with  a  fair  degree  of  accu- 
racy, of  the  numbers  of  some  particular  organism 
(e.  g.y  B.  coli  communis),  present  per  unit,  in  the 
sample  under  examination. 

The  general  principles  underlying  the  bacteriological 
analyses  of  water,  sewage,  air  and  dust,  soil,  milk,  ice 
cream,  meat,  and  other  tinned  stuffs,  as  exemplified 
by  the  methods  used  by  the  author,  are  indicated 
in  the  following  pages,  together  with  the  methods  of 
testing  filters  and  chemical  germicides.  It  is  hoped 
that  the  methods  given  will  be  found  to  be  capable 
of  expansion  and  adaptation  to  any  circumstance  or 
set  of  circumstances  which  may  confront  the  student. 


BACTERIOLOGICAL  EXAMINATION  OF  WATER. 

1.  Quantitative. — 

Collection  of  the  Sample. — As  the  quantity  of  water 
actually  used  for  this  examination  rarely  exceeds  2  c.c., 
the  most  suitable  vessels  for  the  reception  of  the  sample 

316 


BACTERIOLOGICAL   EXAMINATION    OF   WATER.     317 


are  small  glass  bottles,  25  c.c.  capacity,  with  narrow 
necks  and  overhanging  glass  stoppers  (to  prevent 
contamination  of  the  bottle  necks  by  falling  dust). 
These  must  be  carefully  sterilised  in  the  hot-air  steril- 
iser (vide  page  35). 

If  the  sample  is  obtained  from  a  tap  or  pipe,  turn 
on  the  water  and  allow  it  to  run  for  a  few  minutes. 
Remove  the  stopper  from  the  bottle  and  retain  it  in 
the  hand  whilst  the  water  is  allowed  to  run  into  the 
bottle  and  three  parts  fill  it.  Re- 
place the  stopper  and  tie  it  down, 
but  do  not  seal  it. 

If  the  sample  is  obtained  from  a 
stream,  tank,  or  reservoir,  fasten  a 
piece  of  stout  wire  around  the  neck 
of  the  bottle,  remove  the  stopper, 
and  retain  it  in  the  hand.  Then, 
using  the  wire  as  a  handle,  plunge 
the  bottle  into  the  water,  mouth 
downwards,  until  it  is  well  beneath 
the  surface;  then  reverse  it,  allow 
it  to  fill,  and  withdraw  it  from  the 
water.  Pour  out  a  few  cubic  cen- 
timetres of  water  from  the  bottle, 
replace  the  stopper,  and  tie  it  down. 

Or,  use  the  apparatus  designed 
by  v.  Esmarch  (Fig.  159),  in  which 
the  stopper  can  be  removed,  the  bottle  filled,  and  the 
stopper  replaced,  whilst  the  bottle  is  below  the  surface 
of  the  water,  even  at  depths  previously  determined 
upon.  When  the  apparatus  is  taken  out  of  the  water, 
the  small  bottles  are  filled  from  it,  and  packed  in  the 
ice-box  mentioned  below. 

To  prevent  the  multiplication  of  the  bacteria  con- 
tained in  the  water  during  transit  from  the  place  of 
collection  to  the  laboratory,  enclose  the  bottles,  rolled 
in  cotton- wool,  in  a  double- walled  metal  box,  pack  the 


Fig.  159. — Esmarch' s 
collecting  bottle  for  water 
samples. 


BACTERIOLOGICAL   ANALYSES. 

space  between  the  walls  with  pounded  ice,  close  the 
metal  box,  and  place  it  in  a  felt-lined  wooden  case 
(Fig.  1 60).  (It  has  been  shown  that  the  majority  of 
bacteria  will  survive  exposure  to  the  temperature  of 
melting  ice  for  some  days,  while  practically  none  will 
multiply  at  this  temperature.) 

On  reaching  the  laboratory,  the  method  of  examina- 
tion consists  in   adding  measured   quantities   of  the 


.  Fig.  1 60. — Ice-box. 

water  sample  to  several  tubes  of  nutrient  media  pre- 
viously liquefied  by  heat,  pouring  plate  cultivations 
from  each  of  these  tubes,  incubating  at  a  suitable  tem- 
perature, and  finally  counting  the  colonies  which 
make  their  appearance  on  the  plates. 

The  bacteria  present  in  the  water  may  comprise 
not  only  varieties  which  have  their  normal  habitat  in 
the  water  and  will  consequently  be  developed  at  20° 
C.,  but  also  varieties  which  have  been  derived  from, 


BACTERIOLOGICAL    EXAMINATION    OF    WATER.     319 

or  are  pathogenic  for,  the  animal  body,  and  which  will 
only  develop  well  at  a  temperature  of  37°  C.  In 
order  to  demonstrate  the  presence  of  each  of  these 
classes  it  will  be  necessary  to  plant  both  gelatine  and 
agar  plates. 

Further,  the  sample  of  water  may  contain  moulds, 
yeasts,  or  torulae,  and  the  development  of  these  will 
be  best  secured  by  plating  in  wort  gelatine  and  incu- 
bating at  20°  C. 

Apparatus  Required: 

Plate-levelling  stand. 

Case  of  sterile  plates. 

Case  of  sterile  pipettes,  i  c.c.  (in  tenths  of  a  cubic  centi- 
metre) . 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  cubic  centi- 
metre) . 

Case  of  sterile  capsules,  25  c.c.  capacity. 

Tubes  of  nutrient  gelatine. 

Tubes  of  nutrient  agar. 

Tubes  of  wort  gelatine. 

One  250  c.c.  flask  of  sterile  distilled  water. 

Tall  cylinder  containing  2  per  cent,  lysol  solution. 

Bunsen  burner. 

Grease  pencil. 

Water-bath  regulated  at  42°  C. 

METHOD. — 

1.  Arrange    the    plate-levelling    platform    with    its 
water  compartment  filled  with  water,  at  45°  C. 

2.  Number  the  agar  tubes  1,2,  and  3;  the  gelatine 
tubes,  consecutively,  i  to  6,  and  the  wort  tubes,  1,2, 
and  3.     Flame  the  plugs  and  see  that  they  are  not 
adherent  to  the  lips  of  the  tubes. 

3.  Place  the  agar  tubes  in  boiling  water  until  the 
medium  is  melted,  then  transfer  them  to  the  water- 
bath  regulated  at  42°  C.     Liquefy  the  nutrient  gelatine 
and  wort  gelatine  tubes  by  immersing  them  in  the 
same  water-bath. 

4.  Remove  the  bottle  containing  the  water  sample 
from   the   ice-box,   distribute   the   bacterial   contents 


320  BACTERIOLOGICAL   ANALYSES. 

evenly  throughout  the  water  by  shaking,  cut  the 
string  securing  the  stopper,  and  loosen  the  stopper, 
but  do  not  take  it  out. 

5.  Remove  one  of  the  i  c.c.  pipettes  from  the  case, 
holding  it  by  the  plain  portion  of  the  tube.     Pass  the 
graduated  portion  twice  through  the  Bunsen  flame, 
raise  the  bottle  containing  the  water  sample  from  the 
bench  in  the  other  hand,  grasp  the  stopper  as  if  it 
were  a  cotton-wool  plug,  and  remove  it  from  the  bottle 
with  the  hand  holding  the  pipette;  flame  the  mouth 
of  the  bottle. 

6.  Pass  the  pipette  into  the  mouth  of  the  bottle, 
holding  its  point  well  below  the  surface  of  the  water. 
Suck  up  rather  more  than  i  c.c.  into  the  pipette;  empty 
the  pipette  by  blowing.     Now  draw  up  exactly  i  c.c. 
into    the   pipette.     Withdraw   the   pipette   from   the 
bottle,  replace  the  stopper,  and  put  the  bottle  down. 

7.  Take  the  first  melted  agar  tube  in  the  left  hand, 
remove  the  cotton-wool  plug,  and  add  to  its  contents 
0.5  c.c.  of  the  water  sample  from  the  pipette;  replug 
the  tube  and  put  it  down.     In  a  similar  manner  add 
0.3  c.c.  water  to  the  contents  of  the  second  tube,  and 
0.2  c.c.  to  the  contents  of  the  third. 

8.  Drop   the   pipette  into   the  cylinder  containing 
lysol  solution. 

9.  Mix  the  water  sample  with  the  medium  in  each 
tube  in  the  manner  described  under  plate  cultivations ; 
pour  a  plate  from  each  tube.     Label  each  plate  with 
(a)  the  distinctive  name  or  number  of  the  sample,  (b) 
the  quantity  of  water  sample  it  contains,  and  (c)  the 
date. 

10.  Allow  the  plates  to  set,  and  incubate  at  37°  C. 

11.  Empty    the    water    chamber    of    the    levelling 
apparatus  and  refill  it  with  ice- water. 

12.  By  means  of  the  sterile  10  c.c.  pipette  deliver 
9.9  c.c.  sterile  distilled  water  into  a  sterile  capsule. 

13.  Add  o.i  c.c.  of  the  water  sample  to  the  9.9  c.c. 


BACTERIOLOGICAL   EXAMINATION    OF    WATER.     321 

sterile  water  in  the  capsule.     This  will  give  a  dilution 
of  i  in  100. 

14.  Plant  the  six  tubes  of  nutrient  gelatine  in  the 
following  manner:  To  the  first  tube  add  0.5  c.c.  of  the 
water  sample  direct  from  the  bottle;  to  the  second, 
0.3  c.c.;  and  to  the  third,  0.2  c.c.;  and  pour  a  plate 
of  each  tube.     To  the  fourth  tube  add  0.5  c.c.  of  the 
diluted  water  sample  from  the  capsule;  to  the  fifth, 
0.3  c.c.;  and  to  the  sixth,  0.2  c.c.;  and  pour  a  plate 
from   each. 

15.  Label  each  plate  with  the  quantity  of  the  water 
sample  it  contains — that  is,  0.5  c.c.,  0.3  c.c. ,0.2  c.c., 
0.005  c-c->  0.003  c-c->  and  0.002  c.c. 

1 6.  Allow  the  plates  to  set,  and  incubate  at  20°  C. 

17.  To  the  first  tube  of  liquefied  wort  gelatine  add 
0.5  c.c.  water  sample;  to  the  second,  0.3  c.c.;  and  to 
the  third,  2  c.c. 

1 8.  Label  the  plates,  allow  them  to  set,  and  incubate 
at  20°  C. 

19.  Count  the  number  of  colonies  that  have  de- 
veloped upon  the  agar  at  37°  C.   after  twenty-four 
hours'  incubation. 

20.  Note  the  number  of  colonies  present  on  each  of 
the  gelatine  and  wort  gelatine  plates  after  forty-eight 
hours'  incubation. 

21.  Replace  in  the  incubator,  count  again  at  three 
days,  four  days,  and  five  days. 

22.  Calculate  the  number  of  organisms  present  per 
cubic  centimetre  of  the  original  water  from  the  average 
of  the  six  gelatine  plates. 

Method  of  Counting.— The  most  accurate  method 
of  counting  the  colonies  on  each  of  the  plates  is  by 
means  of  either  Jeffer's  or  Fakes'  counting  disc.  Each 
of  these  discs  consists  of  a  piece  of  paper,  upon  which 
is  printed  a  dead  black  disc,  subdivided  by  concentric 
circles  and  radii,  printed  in  white.  In  Jeffer's  counter, 
each  subdivision  has  an  area  of  i  square  centimetre; 

21 


322         BACTERIOLOGICAL  ANALYSES. 


Fig.  161. — Jeffer's  disc. 


Fig.  162. — Fakes'  disc. 


BACTERIOLOGICAL   EXAMINATION   OF   WATER.    323 

in  Fakes'  counter,  radii  divide  the  circle  into  sixteen 
equal  sectors,  and  counting  is  facilitated  by  equidistant 
concentric  circles. 

(a)  In  the  final  counting  of  each  plate,  place  the 
plate  over  the  counting  disc,  and  centre  it,  if  possible, 
making  its  periphery  coincide  with  one  or  other  of  the 
concentric  circles. 

(b)  Remove  the  cover  of  the  plate,  and  by  means  of 
a  hand  lens  count  the  colonies  appearing  in  each  of  the 
sectors  in  turn.     Make  a  note  of  the  number  present 
in  each. 

(c)  If  the  colonies  present  are  fewer  than  500,  the 
entire  plate   should  be  counted.     If,   however,   they 
exceed    this    number,    enumerate    one-half,    or    one- 
quarter  of  the  plate,  or  count  a  sector  here  and  there, 
and  from  these  figures  estimate  the  number  of  colonies 
present  on  the  entire  plate. 

It  will  be  noted  that  the  quantities  of  water  selected 
for  addition  to  each  set  of  tubes  of  nutrient  media 
total  i  c.c.;  consequently  the  plates  in  a  measure 
control  each  other;  that  is,  the  second  and  third 
plates  of  a  series  should  together  contain  as  many 
colonies  as  the  first,  and  the  second  should  contain 
about  half  as  many  more  than  the  third. 

2.  Qualitative  Examination.— 

Collection  of  the  Sample. — The  quantity  of  water 
required  for  this  examination  is  about  2000  c.c.,  and 
the  vessel  usually  chosen  for  its  reception  is  an  ordinary 
blue  glass  Winchester  quart  bottle,  sterilised  in  the 
hot-air  oven,  the  stopper  covered  by  a  piece  of  sterile 
cotton- wool,  and  over  this  a  paper  or  parchment  cap 
fastened  with  string.  The  bottle  may  be  packed  in 
a  wooden  box  or  in  an  ordinary  wicker  case.  The 
method  of  collecting  the  sample  is  identical  with  that 
described  under  the  heading  of  Quantitative  Examina- 
tion; there  is,  however,  not  the  same  imperative 


324         BACTERIOLOGICAL  ANALYSES. 

necessity  to  pack  the  sample  in  ice  for  transmission 
to  the  laboratory. 

Examination. — The  qualitative  bacteriological  ex- 
amination of  water  is  usually  directed  to  the  deter- 
mination of  the  presence  or  absence  of  certain  patho- 
genic organisms,  usually  one  or  more,  but  very  rarely 
all,  of  those  comprised  in  the  following  list : 

I.  B.  coli  communis. 
II.  B.  typhi  abdominalis. 

III.  B.  enteritidis  of  Gaertner. 

IV.  B.  enteritidis  sporogenes. 
V.  Streptococci. 

VI.  Vibrio  cholerae. 
VII.  B.  anthracis. 
VIII.  B.  tetani. 

When  these  are  present  they  are  usually  very  highly 
diluted,  and  it  is  necessary,  before  commencing  the 
examination,  to  adopt  some  means  by  which — 

1.  All  the  bacteria  present  in  the  sample  of  water, 
pathogenic  or  otherwise,   may  be  concentrated  in  a 
small  space. 

2.  The  harmless  non-pathogenic  bacteria  may  be 
destroyed  or  their  growth  inhibited. 

The  first  of  these  objects  is  usually  effected  by 
filtration  of  the  water  sample  through  a  porcelain 
filter  candle,  and  the  subsequent  emulsion  of  the 
bacterial  residue  of  the  original  water  with  a  small 
measured  quantity  of  sterile  bouillon. 

The  second  is  attained  by  so  arranging  the  en- 
vironment (i.  e.,  media,  incubation  temperature,  and 
atmosphere)  as  to  favor  the  growth  of  the  pathogenic 
organisms  at  the  expense  of  the  harmless  saprophytes. 

Apparatus  Required: 

Sterile  Berkfeld  porcelain  filter  candle,  fitted  with  rubber 
washer. 

Rubber  cork  to  fit  the  mouth  of  the  filter  candle,  perfo- 
rated with  one  hole. 

Kitasato  serum  flask,  2000  c.c.  capacity. 


BACTERIOLOGICAL   EXAMINATION   OF   WATER.    325 

Fleuss  air  pump  or  water  force  pump. 

Wulff's  bottle,  fitted  as  wash-bottle,  and  containing  sul- 
phuric acid  (to  act  as  a  safety  valve  between  filter  and 
pump). 

Pressure  tubing,  clamps,  pinch-cock. 

Retort  stand,  with  ring  and  clamp. 


Fig.  163. — Water  filtering  apparatus.  That  portion  of  the  figure  to  the  left 
of  the  vertical  line  is  drawn  to  a  larger  scale  than  that  on  the  right,  in  order 
to  show  details  of  the  force  pump. 


Rubber  cork  for  the  neck  of  Winchester  quart,  perforated 
with  two  holes  and  fitted  with  one  6  cm.  length  of 
straight  glass  tubing,  and  one  V-shaped  piece  of  glass 
tubing,  one  arm  32  cm.  in  length,  the  other  52  cm.,  the 
short  arm  being  plugged  with  cotton-wool.  The  rub- 
ber stopper  must  be  sterilised  by  boiling  and  the  glass 
tubing  by  hot  air,  before  use. 


326 


BACTERIOLOGICAL    ANALYSES. 


Flask  containing  250  c.c.  sterile  broth. 

Test-tube  brush  to  fit  the  lumen  of  the  candle,  enclosed  in 
a  sterile  test-tube  (and  previously  sterilised  by  dry  heat 
or  by  boiling). 

Case  of  sterile  pipettes,  10  c.c.  in  tenths. 

Case  of  sterile  pipettes,  i  c.c.  in  tenths. 

Case  of  sterile  pipettes,  i  c.c.  in  hundredths. 

Tubes  of  various  nutrient  media  (according  to  require- 
ments). 

Twelve  Buchner's  tubes  with  rubber  stoppers. 

Pyrogallic  acid,  10  per  cent,  aqueous  solution. 

Dekanormal  caustic  soda  solution. 

METHOD.— 

i.  Fit  up  the  filtering  apparatus  as  in  the  accom- 
panying diagram  (Fig.  163),  interposing  the  wash- 
bottle  with  sulphuric  acid  between  the 
filter  flask  and  the  force-pump  (in  the 
position  occupied  in  the  diagram  by 
the  central  vertical  line),  and  placing 
another  screw  clamp  on  the  rubber 
tubing  connecting  the  lateral  arm  of 
the  filter  flask  with  the  wash-bottle. 

2.  Filter  the  entire  2000  c.c.  of  water 
through  the  filter  candle. 

3.  When  the  filtration  is  completed, 
screw  up   the  clamps  and   so   occlude 
the  two  pieces  of  pressure  tubing. 

4.  Reverse  the  position  of  the  glass 
tubes  in  the  WulfTs  bottle  so  that  the 
one  nearest  the  air  pump  now  dips  into 
the  sulphuric  acid. 

5.  Slowly  open  the  metal  clamps  and 
allow  air  to  gradually  enter  filter  flask, 
having   first   passed  through  the  acid, 
and  restore  the  pressure. 

6.  Unship  the  apparatus,  remove  the  cork  from  the 
mouth  of  the  candle. 

7.  Pipette  10  c.c.  of  sterile  broth  into  the  interior 
of  the  candle,  and  by  means  of  the  sterile  test-tube 


Fig.   164. — Sterile 
test-tube  brush. 


THE  COU  AND  TYPHOID  GROUPS.       327 

brush  (Fig.  164)  emulsify  the  slimy  residue  which 
lines  the  candle,  with  the  broth. 

The  entire  bacterial  contents  of  the  original  2000  c.c. 
of  water  are  now  suspended  in  10  c.c.  of  broth,  so  that 
i  c.c.  of  the  suspension  is  equivalent,  so  far  as  the 
contained  organisms  are  concerned,  to  200  c.c.  of  the 
original  water. 

Up  to  this  point  the  method  is  identical,  irrespective 
of  the  particular  organism  whose  presence  it  is  desired 
to  demonstrate;  but  from  this  point  onwards  the 
methods  must  be  specially  adapted  to  the  isolation 
of  definite  groups  of  organisms  or  of  individual  bacteria. 

The  Coli  and  Typhoid  Groups.— 

1 .  Number  ten  tubes  of  bile  salt  broth  (vide  page 
1 69) ,  consecutively  from  i  to  10. 

2.  To  each  of  the  tubes  of  bile  salt  broth  add  varying 
quantities  of  the  suspension  by  means  of    suitably 
graduated  sterile  pipettes,  as  follows : 

No.  2  ...  o.oi  c.c.  (equivalent  to       2  c.c.  of  the  original  water  sample). 

No.  3  ...  0.02  "  "  "       4  "  "  "  "  "  " 

No.  4  .    .    .0.05  "  "  "     10  "  "  "  "  "  " 

No.  5  ...  o.i  "  "  "20  "  "  "  "  "  " 

No.  6  ...  0.2  "  "  "     40  "  "  "  "  "  .   " 

No.  7  ...  0.3  "  "  "     60  "  "  " 

No.  8  ...  0.5  "  "  «<   100  "  "  "  " 

No.  9  .    .    .  i.o  "  "  <«  200  c«  '«  "  "  "  " 

No.  10  ...  2.5  "  "  "  500  4<  "  "  "  "  " 

(To  No.  i  should  be  added  i  c.c.  of  the  original 
water  sample  before  the  filtration  is  commenced.1) 

3.  Put  up  each  tube  anaerobically  in  a  Buchner's 
tube  and  incubate  at  42°  C. 

1  If  a  positive  result  is  obtained  when  using  this  method,  it  only  needs  a 
simple  calculation  to  determine  the  smallest  quantity  (down  to  I  c.c.)  of  the 
sample  that  contains  at  least  one  of  the  germs.  For  instance,  if  growth  occurs 
in  all  the  tubes  from  4  to  IO,  and  that  growth  is  subsequently  proved  to  be  due 
to  the  presence  of  the  B.  coli,  then  it  follows  that  at  least  one  colon  bacillus  is 
present  in  every  10  c.c.  of  the  water  sample,  but  not  in  every  4  c.c.  If,  on  the 
other  hand,  the  presence  of  the  B.  coli  can  only  be  proved  in  tube  No.  10,  then 
the  average  number  of  colon  bacilli  present  in  the  sample  is  two  per  litre. 


328  BACTERIOLOGICAL   ANALYSES. 

4.  Examine    after    twenty-four   hours'    incubation. 
Note  in  each  culture  tube: 

(a)  The  presence  or  absence  of  visible  growth. 

(6)  The  reaction  of  the  medium  in  each  tube  as  in- 
dicated by  the  colour  change  (if  any)  the  litmus  has 
undergone. 

(c)  The  presence  or  absence  of  gas  formation  as  in- 
dicated by  froth  on  the  surface  of  the  medium  and 
the  collection  of  gas  in  the  fermentation  tube. 

5.  Replace  those  tubes  which  show  no  signs  of  growth 
in  the  incubator.     Examine  after  another  period  of 
twenty-four  hours  with  reference  to  the  same  points. 

6.  Remove  the   culture   tubes   which  show  visible 
growth  from  the  Buchner's  tubes  and  make  gelatine 
plate  cultivations  whether  gas  production  and  acid 
production  are  present  or  not.     Incubate  at  22°  C. 
for  forty-eight  or  seventy- two  hours. 

7.  Pick  off  coliform  or  typhiform  colonies  and  sub- 
cultivate  in: 

1.  Nutrient  bouillon. 

2.  Glucose  formate  bouillon. 

3.  Glycerine  bouillon. 

4.  Lactose  litmus  bouillon. 

5.  Peptone  water. 

6.  Litmus  milk. 

7.  Glucose  gelatine  stab. 

8.  Lactose  gelatine  stab. 

9.  Maltose  gelatine  stab. 

10.  Neutral  red-agar  stab. 

11.  Nutrient  gelatine  streak. 

12.  Potato. 

8.  Differentiate  by  means  of  the  characters  of  the 
resulting  cultural  reactions  into  members  of  the  coli 
group,  members  of  the  Gaertner  group,  and  members 
of  the  typhoid  group. 

9.  Confirm  these  results  by  testing  the  organisms 
isolated  with   serum   obtained  from  animals   experi- 


THE    COLI    AND   TYPHOID   GROUPS. 


329 


mentally  protected  against  each  of  these  groups  of 
bacteria. 


ANALYSIS  OF  MEMBERS  OF  THE   COLI   AND  TYPHOID  GROUPS. 

Lactose  litmus  bouillon. 


Gas. 

B.  coli  communis  and  its  allies. 

i 

Gas  in  glucose  gelatine. 
Acid  and  coagulation  in  milk. 
General  turbidity  and  indol  in  bouillon. 
Fermentation  in  saccharose  media. 


No  gas 


Gaertner  and  typhoid 
groups. 

Glucose  gelatine. 


Gas. 


Gaertner. 


No  gas. 


Typhoid. 


Litrm 

Acid 
Alkal 
Nocc 

is  milk. 

at  first, 
ine  later, 
•agulation. 

Bot 

Get 
No 
•      Ser 

illon. 

eral  turbidity, 
indol. 
um  reaction. 

Litmus 

Acid. 
No  co 

milk.                   Bou 

Gen 
agulation.            No 
Sen 

illon. 

eral  turbidity, 
indol. 
im  reaction. 

Alternative  Methods. — 
(A)  The  Carbolic  Method: 

1.  Take  ten  tubes  of  carbolised  bouillon  (vide  page 
144)  and  number  them  consecutively  from  i  to  10. 

2.  Inoculate  each  tube  with  a  different  amount  of  the 
suspension,  as  in  the  previous  method. 

3.  Incubate  aerobically  at  37°  C. 

4.  Examine    the    culture    tubes    after    twenty-four 
hours'  incubation. 

5.  From  those  tubes  which  show  signs  of  growth, 


330         BACTERIOLOGICAL  ANALYSES. 

pour  plates  in  the  usual  manner,  using  carbolised 
gelatine  (vide  page  149)  in  place  of  the  ordinary  gela- 
tine, and  incubate  at  20°  C.  for  three,  four,  or  five  days 
as  may  be  necessary. 

6.  Subcultivate  from  any  colonies  that  make  their 
appearance,  and  determine  their  identity  on  the 
lines  laid  down  in  the  previous  method. 

(B)  Parietti's  Method: 

1.  Take    nine    tubes    of    Parietti's    bouillon     (vide 
page    144) — i.  e.,  three  each  of  those  containing  o.i 
c.c.,  0.2  c.c.,  and  0.5  c.c.  of  Parietti's  solution  respec- 
tively.    Mark  plainly  on  the  outside  of  each  tube  the 
quantity  of  Parietti's  solution  it  contains. 

2.  To  each  tube  add  a  different  amount  of  the  original 
water,  or  of  the  suspension,  and  incubate  at  37°  C. 

3.  Examine  the  culture  tubes  after  twenty-four  and 
forty-eight  hours'   incubation,   and  plate  in  gelatine 
from  such  as  have  grown. 

4.  Pick  off  suspicious  colonies,  if  any  such  appear 
on  the  plates,   subcultivate   them  upon  the  various 
media,  and  identify  them. 

(C)  Eisner's  Method:  This  method  simply  consists 
in  substituting  Eisner's  potato  gelatine  (vide  page  164) 
for  ordinary  nutrient  gelatine  in  any  of  the  previously 
mentioned  methods. 

(D)  The  Candle  Method: 

1.  Remove  the  rubber  stopper  from  the  mouth  of 
the  filter  candle,  introduce  10  c.c.  sterile  bouillon  into 
its  interior,  and  emulsify  the  bacterial  sediment;  re- 
plug the  mouth  of  the  candle  with  a  wad  of  sterile 
cotton- wool. 

2.  Remove  the  filter  candle  from  the  filter  flask 
and  insert  it  into  the  mouth  of  a  flask  or  a  glass  cylinder 
containing  sterile  bouillon  sufficient  to  reach  nearly  up 
to  the  rubber  washer  on  the  candle. 

3.  Incubate  for  twenty-four  to  thirty-six  hours  at 
37°  C. 


BACILLUS   ENTERITIDIS    SPOROGENES.  331 

4.  From  the  now  turbid  bouillon  in  the  glass  cylinder 
pour  gelatine  plates  and  incubate  at  20°  C. 

5.  Subcultivate  and  identify  any  suspicious  colonies 
that  appear. 

(The  method  depends  upon  the  assumption  that 
members  of  the  typhoid  and  coli  groups  find  their  way 
through  the  porcelain  filter  from  the  interior  to  the 
surrounding  bouillon  at  a  quicker  rate  than  the  asso- 
ciated bacteria.) 

B.  Enteritidis  Sporogenes.— 

1.  Transfer  5  c.c.  of  the  emulsion  from  the  filter 
candle  to  a  sterile  test-tube  and  plug  carefully. 

2.  Place  the  test-tube  in  the  interior  of  the  benzole 
bath  employed  in  separating  out  spore-bearing  organ- 
isms (vide  page  202),  and  expose  to  a  temperature  of 
80°  C.  for  twenty  minutes. 

3.  Number  ten  tubes  of  litmus  milk  consecutively 
from  i  to  10. 

4.  Remove  the  test-tube  from  the  benzole  bath  and 
shake  well  to  distribute  the  spores  evenly  through 
the  fluid. 

5.  To  each  tube  of  litmus  milk  add  a  measured  quan- 
tity of  the  suspension  corresponding  to  the  amounts 
employed  in  isolating  the  coli  group  (vide  page  327). 

6.  Put  up  each  tube  anaerobically  in  a  Buchner's 
tube  and  incubate  at  37°  C. 

7.  Examine    after    twenty-four    hours'    incubation. 
Note  (if  the  B.  enteritidis  sporogenes  is  present)— 

(a)  Acid   reaction  of  the  medium  as  indicated  by 
the  colour  of  the  litmus  or  its  complete  decolourisation. 

(b)  Presence  of  clotting,  and  the  separation  of  clear 
whey. 

(c)  Presence  of  gas,   as  indicated  by  fissures  and 
bubbles    in    the    coagulum,  and    possibly   masses   of 
coagulum  driven  up  the  tube  almost  to  the  plug. 

8.  Replace  the  tubes  which  show  no  signs  of  growth 
in  the  incubator  for  a  further  period  of  twenty-four 


332         BACTERIOLOGICAL  ANALYSES. 

hours  and  again  examine  with  reference  to  the  same 
points. 

9.  Remove  those  tubes  which  give  evidence  of  growth 
from  the  Buchner's  tubes  and  carefully  pipette  off 
the  whey;  examine  the  whey  microscopically. 

10.  Inoculate  two  guinea-pigs  subcutaneously  with 
0.5  c.c.  of  the  whey  each  and  observe  the  result. 

Streptococci.— 

1.  Melt  ten  tubes  of  nutrient  agar  in  boiling  water 
and  cool  to  42°  C. 

2.  Number  the  tubes  consecutively  from  i  to  10. 

3.  To   each   of   the   tubes  of  liquefied  agar  add   a 
measured  quantity  of  the  emulsion,  corresponding  to 
those  amounts  employed  in  isolating  members  of  the 
coli  group  (vide  page  327). 

4    Pour  plates   in   the  usual  manner  and  incubate 
aerobically  at  37°  C.  for  twenty-four  hours. 

5.  Examine  the  plates  carefully,  pick  off  suspicious- 
looking  colonies,  and  subcultivate  in  broth. 

6.  If  the  resulting  growth  appears,  microscopically, 
to  be  composed  of  streptococci,  subcultivate  on  the 
various  media  and  identify. 

Vibrio  Cholerse.— 

1 .  Number  ten  tubes  of  peptone  water  consecutively 
from  i  to  10. 

2.  To  each  of  the  tubes  of  peptone  water  add  a  mea- 
sured  quantity  of  the  suspension,   corresponding  to 
those  amounts  employed  in  isolating  the  members  of 
the  coli  group  (vide  page  327). 

3.  Incubate   aerobically  at   37°   C.  for  twenty-four 
hours.     Examine  the  tubes  carefully  for  visible  growth, 
especially  delicate  pellicle  formation,  which  if  present 
should  be  examined  microscopically  for  vibrios. 

4.  Inoculate  fresh  tubes  of  peptone  water  from  such 
of  the  tubes  as  exhibit  pellicle  formation, — from  the 
pellicle   itself, — and   incubate   at   37°  C.  for   twenty- 
four  hours. 


BACILLUS   ANTHRACIS — BACILLUS   TETANI.       333 

5.  Prepare  gelatine  and  agar  plates  in  the  usual  way 
from  such  of  these  tubes  as  show  pellicle  formation. 

6.  Pick  off  from  the  plates  any  colonies  resembling 
those  of  the  Vibrio  cholerae  and  subcultivate  upon  all 
the  ordinary  laboratory  media. 

7.  Test  the  vibrio  isolated  against  the  serum  of  an 
animal  immunised  to  the  Vibrio  cholerae. 

B.  Anthracis.— 

1.  Transfer  5  c.c.  of  the  emulsion  from  the  filter 
candle  to  a  sterile  test-tube  and  plug  carefully. 

2.  Place  the  test-tube  in  the  interior  of  the  benzole 
bath  employed  in  separating  out  spore-bearing  organ- 
isms  (vide  page    202),  and  expose  to  a  temperature 
of  80°  C.  for  twenty  minutes. 

3.  Melt  three  tubes  of  nutrient  agar  in  boiling  water 
and  cool  to  42°  C. 

4.  Number  the  tubes  i,  2,  and  3.     To  No.   i    add 
0.2  c.c.,  to  No.  2  add  0.3  c.c.,  and  to  No.  3  add  0.5  c.c. 
of  the  suspension,  and  pour  plates  therefrom. 

5.  Incubate    at    37°    C.    for    twenty-four   or   forty- 
eight  hours. 

6.  Pick  off  any  colonies  resembling  those  of  anthrax 
and  subcultivate  on  all  the  ordinary  laboratory  media. 

7.  Inoculate  a  young  white  rat  subcutaneously  (on 
the  inner  aspect  of  one  of  the  hind  legs)  with  two 
loopfuls   of  the  cultivation   on   agar,  emulsified  with 
i   c.c.   sterile  bouillon.     Observe  during  life,   and,  if 
the  animal  succumbs,  make  a  complete  post-mortem 
examination. 

B.  Tetani.- 

1.  Proceed  as  detailed  above  in  steps  i  and  2  for 
the  isolation  of  the  B.  anthracis. 

2.  Add  i  c.c.  of  the  suspension  to  each  of  three  tubes 
of  glucose  formate  broth,  and  incubate  anaerobically 
in  Buchner's  tubes  at  37°  C. 

3.  From  such  of  the  tubes  as  show  visible  growth 
(with  or  without  the  production  of  gas)  after  twenty- 


334  BACTERIOLOGICAL   ANALYSES. 

four  hours'  incubation  inoculate  guinea-pigs,  subcu- 
taneously  (under  the  skin  of  the  abdomen),  using  o.i 
c.c.  of  the  bouillon  cultivation  as  a  dose.  Observe 
carefully  during  life,  arid,  if  death  occurs,  make  a 
complete  post-mortem  examination. 

4.  From  the  same  tubes  pour  agar  plates  and  in- 
cubate anaerobically  in  Bulloch's  apparatus,  at  37°  C. 

5.  Subcultivate  suspicious  colonies  on  the  various 
media,  incubate  anaerobically,  making  control  cultiva- 
tions on  glucose  formate  agar,  stab  and  streak,  to 
incubate  aerobically. 


EXAMINATION  OF    SEWAGE  AND   SEWAGE  EFFLUENTS. 

Quantitative. — 

Collection  of  the  Sample. — As  only  small  quantities 
of  material  are  needed,  the  samples  should  be  collected 
in  a  manner  similar  to  that  described  under  water  for 
quantitative  examination  and  transmitted  in  the  ice 
apparatus  used  in  packing  those  samples. 

Apparatus  Required. — As  for  water  (vide  page  319), 
substituting  gelatine  tubes  for  the  agars  and  the 
wort  gelatines. 

METHOD. — 

1.  Arrange  four  sterile  capsules  in  a  row  and  number 
them  I,  II,  III,  IV. 

2.  Pipette  9  c.c.  sterile  bouillon  into  capsule  No.  I. 

3.  Pipette  9.9  c.c.  sterile  bouillon  into  capsules  II, 
III,  and  IV. 

4.  Add  i  c.c.  of  the  sewage  to  capsule  No.  I  by  means 
of  a  sterile  pipette,  and  mix  thoroughly. 

5.  Take  a  fresh  sterile  pipette  and  transfer  o.i  c.c. 
of  the  mixture  from  No.  I  to  No.  II  and  mix  thor- 
oughly. 

6.  In  like  manner  transfer  o.i  c.c.  from   No.  II   to 
No.  Ill,  and  then  o.i  c.c.  from  No.  Ill  to  No.  IV. 


EXAMINATION   OF   AIR.  335 

Now  I  c.c.  of  dilution  No.       I  contains  O.  I  c.c.  of  the  original  sewage. 

I     "       "          «  "          II  "          O.OOI  "  "  "  " 

I     "       "          *<  ««       HI  "  0.00001  "  "  "  " 

!     "       «          «  «        IV  "          0.0000001      "          "  "  " 

7.  Pour  a  set  of  gelatine  plates  from  the  contents 
of  each  capsule,  three  plates  in  a  set,  and  containing 
respectively  0.2,    0.3,    and   0.5  c.c.  of   the    dilution. 
Label  carefully;  incubate  at  20°  C.  for  three,  four,  or 
five  days. 

8.  Enumerate  the  organisms  present  in  those  sets 
of   plates  which  have  not  liquefied,  probably  those 
from  dilution  III  or  IV,  and  calculate  therefrom  the 
number  present  per  cubic  centimetre  of  the  original 
sample  of  sewage. 

Qualitative. — The  qualitative  examination  of  sewage 
is  but  rarely  required.  When  necessary,  however,  it 
is  conducted  on  lines  similar  to  those  indicated  under 
the  corresponding  section  of  water  examination. 


EXAMINATION  OF   AIR. 

Quantitative. — 

Apparatus  Required: 

Aspirator  bottle,  10  litres  capacity,  fitted  with  a  delivery 
tube,  and  having  its  mouth  closed  by  a  perforated  rubber 
stopper,  through  which  passes  a  short  length  of  glass 
tubing. 

Brlenmeyer  flask,  250  c.c.  capacity  (having  a  wide  mouth 
properly  plugged  with  wool),  containing  50  c.c.  sterile 
bouillon. 

Rubber  stopper  to  fit  the  mouth  of  the  flask,  perforated 
with  two  holes,  and  fitted  as  follows : 

Take  a  15  cm.  length  of  glass  tubing  and  bend  up  3 
cm.  at  either  end  at  right  angles  to  the  main  length  of 
tubing.  Pass  one  of  the  bent  ends  through  one  of  the 
perforations  in  the  stopper ;  plug  the  opposite  end  with 
cotton- wool. 

Take  a  glass  funnel  5  or  6  cm.  diameter  with  a  stem 
12  cm.  in  length  and  bend  the  stem  close  up  to  the  apex 
of  the  funnel,  in  a  gentle  curve  through  a  quarter  of  a 
circle ;  pass  the  long  stem  through  the  other  perforation 
in  the  rubber  stopper. 


336 


BACTERIOLOGICAL  ANALYSES. 


Rubber  tubing. 

Screw  clamps  and  spring  clips,  for  tubing. 
Water  steriliser. 
Retort  stand  and  clamps. 

Apparatus  for  plating  (as  for  enumeration  of  water  organ- 
isms, -vide  page  319). 

METHOD.— 

i.  Fill  10  litres  of  water  into  the  aspirating  bottle 
and  attach  a  piece  of  rubber  tubing  with  a  screw 
clamp  to  the  delivery  tube.  Regulate  the  screw  clamp, 


Fig.  165. — Arrangement  of  apparatus  for  air  analysis. 

by  actual  experiment,  so  that  the  tube  delivers  i  c.c. 
of  water  every  second. 

At  this  rate  the  aspirator  bottle  will  empty  itself 
in  just  under  three  hours.  Occlude  the  rubber  tube 
below  the  screw  clamp  by  means  of  a  spring  clip, 
and  make  up  the  contents  of  the  aspirator  bottle  to 
10  litres  again. 

2.  Sterilise  the  fitted  rubber  cork,  with  its  funnel 


EXAMINATION  OX  AIR.  337 

and  tubing,  by  boiling  in  the  water  steriliser  for  ten 
minutes. 

3.  Remove  the  cotton- wool  plug  from  the  flask,  and 
replace  it  by  the  rubber  stopper  with  its  fittings.     Make 
sure  that  the  end  of  the  stem  of  the  funnel  is  immersed 
in  the  bouillon. 

4.  Place  the  flask  in  a  glass  or  metal  vessel  and  pack 
it  round  with  pounded  ice.     Arrange  the  flask  with  its 
ice  casing  just  above  the  neck  of  the  aspirator  bottle. 

5.  Connect  up  the  free  end  of  the  glass  tube  from  the 
flask — after  removing  the  cotton- wool  plug — with  the 
air-entry  tube  in  the  mouth  of  the  aspirating  bottle. 

6.  Remove  the  spring  clip  from  the  rubber  tube,  and 
allow  the  water  to  run. 

Replenish  the  ice  from  time  to  time  if  necessary. 
(In  emptying  itself  the  aspirator  bottle  will  aspirate 
10  litres  of  air  slowly  through  the  broth  in  the  Brlen- 
meyer  flask.) 

7.  When  the  aspiration  is  completed,  disconnect  the 
flask  and  remove  it  from  its  ice  packing. 

8.  Liquefy  three  tubes  of  nutrient  gelatine  and  add 
to  them  0.5  c.c.,  0.3  c.c.,  and  0.2  c.c.,  respectively,  of 
the  broth  from  the  flask,  by  means  of  a  sterile  gradu- 
ated  pipette,  as   in  the  quantitative   examination  of 
water.     Pour  plates. 

9.  Pour  a  second  similar  set  of  gelatine  plates. 

10.  Incubate  both  sets  of  plates  at  20°  C. 

11.  Enumerate  the  colonies  present  in  the  two  sets 
of  gelatine  plates  after  three,  four,  or  five  days  and 
average  the  results  from  the  numbers  so  obtained; 
estimate  the  number  of  micro-organisms  present  in 
i  c.c.,  and  then  in  the  50  c.c.  of  broth  in  the  flask. 

12.  The  result  of  air  examination  is  usually  expressed 
as  the  number  of  bacteria  present  per  cubic  metre 
(i.  e.t  kilolitre)  of  air;  and  as  the  number  of  organisms 
present  in  the  50  c.c.  bouillon  only  represent  those 


22 


338  BACTERIOLOGICAL  ANALYSES. 

contained  in  10  litres  of  air,  the  resulting  figure  must 
be  multiplied  by  1000. 
Qualitative. — 

1.  Proceed  exactly  as  in  the  quantitative  examina- 
tion of  air  (vide  supra),  steps  i  to  10. 

2.  Pour  plates  of  wort  agar  with  similar  quantities 
of  the  air-infected  bouillon,  and  incubate  at  37°  C. 

3.  Pour  plates  of  nutrient  agar  with  similar  quan- 
tities of  the  bouillon  and  incubate  at  37°  C. 

4.  Pour  similar  plates  of  wort  gelatine  and  incubate 
at  20°  C. 

5.  Pick  off  the  individual  colonies  that  appear  in 
the  several  plates,  subcultivate  them  on  the  various 
media,  and  identify  them. 


EXAMINATION  OF  SOIL. 

Collection  of  Sample. — A  small  copper  capsule  6  cm. 
high  by  6  cm.  diameter,  with  "pull-off"  cap  secured 
by  a  bayonet  catch,  previously  sterilised  in  the  hot- 
air  oven,  is  the  most  convenient  receptacle  for  samples 
of  soil. 

The  instrument  used  for  the  actual  removal  of  the 
soil  from  its  natural  position  will  vary  according  to 


Fig.  1 66. — Soil  scoop. 

whether  we  require  surface  samples  or  soil  from  vary- 
ing depths.  In  the  first  case,  use  an  iron  scoop,  shaped 
like  a  shoe  horn,  but  provided  with  a  sharp  spine 
(Fig.  1 66).  This  is  wrapped  in  asbestos  cloth  and 
sterilised  in  the  hot-air  oven.  When  removed  from 
the  oven,  wrap  in  a  piece  of  oiled  paper,  silk,  or  gutta- 


EXAMINATION   OF   SOIL. 


339 


percha  tissue,  secured  with  string,  as  a  further  protec- 
tion against  contamination. 

On  reaching  the  spot  whence  the  samples  are  to  be 
taken,  the  coverings  of  the  scoop  are  removed,  and  the 
asbestos  cloth  employed  to  brush  away  loose  stones 
and  debris  from  the  selected  area.  The  surface  soil 
is  then  broken  up  with  the  point  of  the  scoop,  scraped 
up  and  collected  in  the  body  of  the  scoop,  and  trans- 
ferred to  the  sterile  cap- 
sule for  transmission. 

If  it  is  desired  to  ob- 
tain samples  of  the  earth 
from  varying  depths, 
some  form  of  borer,  such 
as  that  designed  by 
Fraenkel  (sterilised  in  a 
manner  similar  to  that 
adopted  for  the  scoop), 
must  be  employed  for 
the  purpose  (Fig.  167). 

Quantitative. — Four 
distinct  investigations 
are  included  in  the  com- 
plete quantitative  bac- 
teriological examination 
of  the  soil: 

1 .  The  enumeration  of 
the  aerobic  organisms. 

2.  The  enumeration  of 
the  spores  of  aerobes. 

3.  The  enumeration  of  the  anaerobic  organisms  (in- 
cluding the  facultative  anaerobes). 

4.  The  enumeration  of  the  spores  of  anaerobes. 
Further,  by  a  combination  of  the  results  of  the  first 

and  second,   and  of  the  third  and  fourth  of  these, 
the  ratio  of  spores  to  vegetative  forms  is  obtained. 


V 

Fig.  167. — Fraenkel' s  borer. 


340         BACTERIOLOGICAL  ANALYSES. 

Apparatus  Required: 

Case  of  sterile  capsules  (25  c.c.  capacity). 

Case  of  sterile  graduated  pipettes,  10  c.c.  (in  tenths  of  a 

cubic  centimetre). 
Case  of  sterile  graduated  pipettes,   i  c.c.  (in  tenths  of  a 

cubic  centimetre). 

Flask  containing  250  c.c.  sterile  bouillon. 
Tall  cylinder  containing  2  per  cent,  lysol  solution. 
Plate-levelling  stand. 
12  sterile  plates. 
Tubes  of  nutrient  gelatine. 
Tubes  of  wort  gelatine. 
Tubes  of  nutrient  agar. 
Tubes  of  glucose  formate  gelatine. 
Tubes  of  glucose  formate  agar. 
Water-bath  regulated  at  42°  C. 
Bunsen  burner. 
Grease  pencil. 

Sterile  mortar  and  pestle  (agate). 

Sterile  wide-mouthed  Erlenmeyer  flask  (500  c.c.  capacity). 
Sterile  metal  funnel  with  short  wide  bore  delivery  tube  to 

just  fit  mouth  of  flask. 

Solid  rubber  stopper  to  fit  the  flask  (sterilised  by  boiling). 
Pair  of  scales. 
Counterpoise  (Fig.  88). 
Sterile  metal  (nickel)  spoon  or  spatula. 
Fractional  steriliser  (Fig.  113). 


METHOD. — 

1.  Arrange  four  sterile  capsules  numbered  I,  II,  III, 
and  IV;  pipette  9  c.c.  sterile  bouillon  into  the  first 
capsule,  and  9.9  c.c.  into  each  of  the  remaining  three. 

2.  Pipette  100  c.c.  sterile  bouillon  into  the  Krlen- 
meyer flask. 

3.  Remove  the  cotton- wool  plug  from  the  flask  and 
replace  it  by  the  sterile  funnel. 

4.  Place  flask  and  funnel  on  one  pan  of  the  scales, 
and  counterpoise  accurately. 

5.  Empty  the  sample  of  soil  into  the  mortar  and 
triturate  thoroughly. 

6.  By  means  of  the  sterile  spatula  add  10  grammes 
of  the  earth  sample  to  the  bouillon  in  the  flask. 

The  final  results  will  be  more  reliable  if  steps  2,  3,  4, 


EXAMINATION   OF  SOIL.  341 

and  5  are  performed  under  a  hood — to  protect  from 
falling  dust,  etc. 

7.  Remove  the  funnel  from  the  mouth  of  the  flask; 
replace  it  by  the  rubber  stopper  and  shake  vigourously ; 
then  allow  the  solid  particles  to  settle  for  about  a 
minute. 

8.  Pipette  off   i   c.c.   of  the  supernatant  bouillon, 
termed  the  "soil  water,"  and  add  it  to  the  contents 
of  capsule  I ;  mix  thoroughly. 

9.  Remove  o.  i  c.c.  of  the  infected  bouillon   from 
capsule  I  and  add  it  to  capsule  II,  and  mix. 

10.  In  like  manner  add  o.i   c.c.  of  the  contents  of 
capsule   II   to   capsule   III   and   then  o.i  c.c.  of  the 
contents  of  capsule  III  to  capsule  IV. 

Then  I  c.c.  fluid  from  capsule     I  contains  soil  water  from  .01  gm.  earth. 

I    «      "       "          «         II       "         "       "        "     .0001  " 

I    «      «       ««         "       III      "         "       "       "     .oooooi       "       " 
I    «      «       "         «        IV      "         "       "       "     .oooooooi  "       " 

(A)  Aerobes  (Vegetative  Forms  and  Spores). — 

11.  Pour  a  set  of  gelatine  plates  from  the  contents 
of  each  capsule — two  plates  in  a  set,  and  containing 
respectively  o.i   c.c.  and  0.3  c.c.  of   the  diluted  soil 
water.     Label  and  incubate. 

12.  Pour  similar  sets  of  wort  gelatine  plates  from  the 
contents  of  capsule  II  and  III,  label,  and  incubate  at 
20°  C. 

13.  Pour  similar  sets  of  agar  plates  from  the  contents 
of  capsules  II  and  III;  label  and  incubate  at  37°  C. 

14.  "Count"  the  plates  after  incubation  for  three, 
four,  or  five  days,  and  from  the  figures  thus  obtained 
estimate — 

(a)  The  number  of  aerobic  micro-organisms  present 
per  gramme  of  the  soil. 

(b)  The  number  of  yeasts  and  moulds  present  per 
gramme  of  the  soil. 

(c)  The  number  of  aerobic  organisms  "growing  at 
37°  C."  present  per  gramme  of  the  soil. 


342  BACTERIOLOGICAL  ANALYSES. 

(B)  Anaerobes  (Vegetative  Forms  and  Spores). — 

15.  Pour  similar  sets  of  plates  in  glucose  formate 
gelatine  and  agar  and  incubate  in  Bulloch's  anaerobic 
apparatus. 

(C)  Aerobes  and  Anaerobes  (Spores  Only}.— 

1 6.  Pipette  5  c.c.  soil  water  into  a  sterile  tube. 

17.  Place  in  the  differential  steriliser  at  80°  C.  for 
ten  minutes. 

1 8.  Pour  two  sets  of  four  gelatine  plates  containing 
o.i,  0.2,  0.5,  and  i  c.c.  respectively  of  the  soil  water; 
label  and  incubate  at  20°  C.,  one  set  aerobically,  the 
other  anaerobically  in  Bulloch's  apparatus. 

19.  "Count"  the  plates  (delay  the  enumeration  as 
long  as  possible)  and  estimate  the  number  of  spores 
of   aerobes   and   anaerobes   respectively   present   per 
gramme  of  the  soil. 

20.  Calculate    the    ratio    existing    between    spores 
alone  and  the  total  number  of  organisms. 

Qualitative  Examination. — The  qualitative  examina- 
tion of  soil  is  usually  directed  to  the  detection  of  one 
or  more  of  the  following : 

I.  Members  of  the  coli  or  typhoid  group. 
II.  Bacillus  enteritidis  sporogenes. 

III.  Streptococci. 

IV.  Bacillus  anthracis. 
V.  Bacillus  tetani. 

VI.  Bacillus  oedematis  maligni. 
VII.  The  nitrous  organisms. 
VIII.  The  nitric  organisms. 

1.  Transfer   the   remainder   of   the   soil    water    (88 
c.c.)  to  a  sterile  Erlenmeyer  flask  by  means  of  a  sterile 
syphon. 

2.  Fix  up  the  filtering  apparatus  as  for  the  qualita- 
tive examination  of  water,  and  filter  the  soil  water. 

3.  Suspend  the  bacterial  residue  in   5   c.c.    sterile 
bouillon  (technique  similar  to  that  described  for  the 
water  sample). 


EXAMINATION   OF   SOU,.  343 

Every  cubic  centimetre  of  suspension  now  contains 
the  soil  water  from  nearly  i  gramme  of  earth. 

The  methods  up  to  this  point  are  identical  no  matter 
which  organism  or  group  of  organisms  it  is  desired 
to  isolate;  but  from  this  stage  onwards  the  process  is 
varied  slightly  for  each  particular  bacterium. 

The  Coli  Group.— 

Bacillus  Enteritidis  Sporogenes. — 

Bacillus  Anthracis.— 

Bacillus  Tetani.— 

The  methods  adopted  for  the  isolation  of  these 
organisms  are  identical  with  those  already  described 
under  water  (page  327  et  seq.). 

Bacillus  (Edematis  Maligni. — Method  precisely  sim- 
ilar to  that  employed  for  the  B.  tetani. 

The  Nitrous  Organisms.— 

The  Nitric  Organisms.— 

i.  Take  six  tubes  of  Warrington's  solution  (vide 
page  172)  and  number  them  consecutively  from  i  to  6. 

2.     To  tube  No.    i   add  o.  i  c.c.   of  the  suspension. 

«       «       «     2       c    02     «     «     «  i 

"       "       "     3 
«       «       «     4 

"      "      '"     5 

"      "       «     6 


0-5 
i.o 

2.5 


Label  and  incubate  at  30°  C. 

3.  Examine  after  twenty-four  and  forty-eight  hours' 
incubation.     From   those   tubes   that   show   signs   of 
growth  make   subcultivations  in  fresh   tubes   of  the 
same  medium  and  incubate  at  30°  C. 

4.  Make  further  subcultivations  from  such  of  those 
tubes  as  show  growth,  and  again  incubate. 

5.  If  growth  occurs  in  these  subcultures,  make  sur- 
face smears  on  plates  of  Winogradsky's  silicate  jelly 
(vide  page  172). 

6.  Pick  off  such  colonies  as  make  their  appearance 
and  subcultivate  in  each  of  these  two  media. 


344  BACTERIOLOGICAL   ANALYSES. 

EXAMINATION  OF  MILK. 

Quantitative. — 

Collection  of  Sample. — "  One-cow"  milk,  if  taken 
from  the  apparently  healthy  animal  (that  is,  an  animal 
without  any  obvious  lesion  of  the  udder  or  teats) 
with  ordinary  precautions  as  to  cleanliness,  avoidance 
of  dust,  etc.,  contains  but  few  organisms,  and  may 
be  received  directly  into  small  sterile  bottles  (similar 
to  those  referred  to  under  the  collection  of  water  for 
quantitative  examination),  packed  in  the  ice-box  for 
transmission,  and  dealt  with  in  precisely  the  same 
manner  as  an  ordinary  water  sample.  In  dealing  with 
one-cow  milk,  from  a  suspected,  or  an  obviously  dis- 
eased animal,  a  complete  analysis  should  include  the 
examination  (both  qualitative  and  quantitative)  of 
samples  of  (a)  fore-milk,  (b)  mid-milk,  (c)  strippings, 
and,  if  possible,  from  each  quarter  of  the  udder,  and 
the  specimen  should  then  be  collected  as  described 
for  mixed  milk. 

"Mixed"  milk,  on  the  other  hand,  by  the  time  it 
leaves  the  retailer's  hands,  usually  contains  as  many 
micro-organisms  as  an  equal  volume  of  sewage,  and  it 
becomes  necessary  to  adopt  special  methods  of  collec- 
tion, and,  when  collected,  to  estimate  the  number  of 
its  contained  bacteria  by  the  methods  employed  in 
the  examination  of  sewage. 

The  apparatus  used  for  the  collection  of  a  retail 
mixed  milk  sample  consists  of  a  cylindrical  copper  case, 
1 6  cm.  high  and  9  cm.  in  diameter,  provided  with  a 
"pull-off"  lid,  containing  a  milk  dipper,  also  made  of 
copper;  and  inside  this,  again,  a  wide-mouthed,  stop- 
pered glass  bottle  of  200  c.c.  capacity  (about  14  cm. 
high  by  7  cm.  diameter),  having  a  tablet  for  notes, 
sand-blasted  on  the  side.  The  copper  cylinder  and  its 
contents,  secured  from  shaking  by  packing  with  cot- 
ton-wool, are  sterilised  in  the  hot-air  oven. 


EXAMINATION   OF   MILK. 


345 


When  collecting  a  sample, 

1.  Remove  the  cap  from  the  cylinder. 

2.  Draw  out  the  cotton-wool. 

3.  Lift  out  the  bottle  and  dipper  together. 

4.  Receive  the  milk  in  the  sterile  dipper,  and  pour  it 
directly  into  the  sterile  bottle. 

5.  Enter  the  particulars  necessary  for  the  identi- 
fication of  the  specimen,  on  the  tablet,  with  a  lead 
pencil,  or  pen  and  ink. 

6.  Repack  the  apparatus. 


Fig.  1 68. — Milk-collecting  bottle  and  dipper. 

Four  such  bottles  should  be  filled,  so  as  to  give  a 
total  of  about  800  c.c.  milk  for  examination.  The  four 
cases  may  be  packed  in  an  ice-box  similar  to,  but 
larger  than,  that  used  for  water  specimens. 

Apparatus  Required: 

Case  of  sterile  capsules  (25  c.c.  capacity). 
Case  of  sterile  graduated  pipettes,  10  c.c.  (in  tenths  of  a 
cubic  centimetre). 


346  BACTERIOLOGICAL   ANALYSES. 

Case  of  sterile  graduated  pipettes,  i  c.c.  (in  tenths  of  a 

cubic  centimetre). 

Flask  containing  250  c.c.  sterile  bouillon. 
Tall  cylinder  containing  2  per  cent,  lysol  solution. 
Plate-levelling  stand. 
Case  of  sterile  plates. 

Tubes  nutrient  gelatine  agar  (-J- 10  reaction). 
Tubes  of  wort  gelatine. 
Tubes  of  nutrient  agar  (+10  reaction). 
Water-bath  regulated  at  42°  C. 
Bunsen  burner. 
Grease  pencil. 

METHOD.— 

1.  Arrange  four  sterile  capsules  in  a  row;  number 
them  I,  II,  III,  and  IV. 

2.  Fill  9  c.c.  sterile  bouillon  into  the  first,  and  9.9 
c.c.  bouillon  into  each  of  the  three  remaining  capsules. 

3.  Remove  i  c.c.  milk  from  one  of  the  bottles  by 
means   of  a  sterile,  pipette  and  dilute  it  exactly  as 
described  for  sewage  (vide  page  334). 

Then  I  c.c.  of  dilution       I  contains  O.I  c.c.  milk  sample. 

I    "     «        •«  II        "        o.ooi  "       " 

i    "    "        "        III        "       o.ooooi  "      "         " 

i    «    ««        "        IV       "       o.ooooooi  "      "         " 

4.  Melt  the  gelatine   agar  and  the   agar   tubes   in 
boiling  water;  then  transfer  to  the  water-bath  and 
cool  them  down  to  42°  C. 

5.  Number  the  gelatine  agar  tubes  consecutively  i 
to  8. 

6. .To  the  first  three  tubes  add  0.2  c.c.,  0.3  c.c.,  and 
0.5  c.c.  respectively  of  the  diluted  milk  from  capsule 
IV. 

7.  To  the  second   set   of  three  tubes   add  similar 
quantities  of  the  diluted  milk  from  capsule  III. 

8.  To  the  two  remaining  tubes  add  o.i  c.c.  and  0.2 
c.c.  of  the  diluted  milk  from  capsule  II. 

9.  Pour  plates  from  the  eight  gelatine  agar  tubes; 
label,  and  incubate  at  28°  C.  (or  30°  C.). 

10.  Liquefy  three  wort  gelatine  tubes  and  to  them 


EXAMINATION   OF  MILK.  347 

add  o.i  c.c.  of  the  diluted  milk  from  capsules  I,  II, 
and  III  respectively. 

11.  Pour  plates  from  the  wort  gelatine;  label,  and 
incubate  at  20°  C. 

12.  Add  to  each  of  three  agar  tubes  o.i  c.c.  of  the, 
diluted  milk  from  capsule  II,  III,  and  IV. 

13.  Pour  plates  from  the  agar  tubes;  label,  and  in- 
cubate at  37°  C. 

14.  After  twenty-four  and  forty-eight  hours'  incu- 
bation,   "  count"    the   agar   plates   and   estimate   the 
number   of   "organisms   growing   at   37°   C."   present 
per  cubic  centimetre  of  the  sample  of  milk. 

15.  After    three,    four,    or    five    days'    incubation, 
"count"  the  gelatine  agar  plates  and  estimate  there- 
from the  total  number  of  organisms  present  per  cubic 
centimetre  of  the  sample  of  milk. 

1 6.  After    a    similar    interval    "count"    the    wort 
gelatine  plates  and  estimate  the  number  of  moulds 
and  yeasts  present  per  cubic  centimetre  of  the  sample 
of  milk. 

Qualitative. — The  qualitative  bacteriological  exam- 
ination of  milk  is  chiefly  directed  to  the  detection  of  the 
presence  of  one  or  more  of  the  following  pathogenic 
bacteria : 

I.  Members  of  the  typhi  and  coli  groups. 
II.  Bacillus  enteritidis  of  Gaertner. 

III.  Bacillus  enteritidis  sporogenes. 

IV.  Vibrio  cholerae.  *«, 
V.  Bacillus  diphtheriae. 

~  VI.  Bacillus  tuberculosis. 

VII.  Streptococcus  pyogenes  longus. 

VIII.  Staphylococcus  pyogenes  aureus. 

Of  these,  the  first  six  occur  as  accidental  contamina- 
tions (the  vehicle  of  transmission  in  the  case  of  the 
first  five  usually  being  water),  while  the  last  three  are 
usually  derived  directly  from  the  cow. 

In  milk,  as  in  water,  the  first  essential  is  the  con- 


34-8  BACTERIOLOGICAL   ANALYSES. 

centration  of  the  bacterial  contents  of  a  large  volume 
of  the  sample  into  a  small  compass.  In  this  process, 
however,  thorough  centrifugalisation  is  substituted  for 
nitration. 

Apparatus  Required: 

,  A  centrifugal  machine.  This  machine,  to  be  of  real  ser- 
vice in  the  bacteriological  examination  of  milk,  must 
conform  to  the  following  requirements : 

1.  The  centrifugal  machine  must  be  of  such  size,  and 

should  carry  tubes  or  bottles  of  such  capacity,  as 
to  enable  from  250  to  500  c.c.  of  milk  to  be 
manipulated  at  one  time. 

2.  The  rate  of  centrifugalisation  should  be  from  2500 

to  3000  revolutions  per  minute. 

3.  The  portion  of  the  machine  destined  to  carry  the 

tubes  should  be  a  metal  disc,  of  sufficient  weight 
to  ensure  good  "flank"  movement,  continuing 
over  a  considerable  period  of  time.  In  other 
words,  the  machine  should  run  down  very 
gradually  and  slowly  after  the  motive  power  is 
removed,  thus  obviating  any  disturbance  of  the 
relative  positions  of  particulate  matter  in  the 
solution  that  is  being  centrifugalised. 

4.  The  machine  should  preferably  be  driven  by  elec- 

tricity, or  by  power,  but  in  the  case  of  hand- 
driven  machines — 

(a)  The  gearing  should  be  so  arranged  that 

the  requisite  speed  is  obtained  by  not 
more  than  forty  or  fifty  revolutions  of 
the  crank  handle  per  minute,  so  that  it 
may  be  maintained  for  periods  of  twenty 
or  thirty  minutes  without  undue  exer- 
tion. 

(b)  The  handle  employed  should  be  provided 

with  a  special  fastening  (e.  g.,  a  clutch 
similar  to  that  employed  for  the  free 
wheel  of  a  bicycle),  or  should  be  readily 
detachable  so  that,  on  ceasing  to  turn, 
the  handle  should  not,  by  its  weight  and 
air  resistance,  act  as  a  brake  and  stop 
the  machine  too  suddenly. 
One  of  the  few  satisfactory  machines  of  this  class  is 

shown  in  figure  169. 

y  Sterile  centrifugal  tubes,  of  some  60-70  c.c.  capacity, 
tapering  to  a  point  at  the  closed  end,  plugged  with 
cotton- wool. 


EXAMINATION   OF   MII^K.  349 

u   Sterilised  cork  borer. 

t/  Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  cubic  centi- 
metre) . 

Case  of  sterile  pipettes,  i  c.c.  (in  tenths  of  a  cubic  centi- 
metre). 
Flask  of  sterile  bouillon. 


Fig.  169. — Electrically  driven  centrifugal  machine. 

METHOD.— 

1.  Fill  the  milk  sample  into  the  tubes,  and  replace 
the  cotton-wool  plugs  by  solid  rubber  stoppers  (steril- 
ised by  boiling),  and  fit   the  tubes  in  the  centrifugal 
machine. 

2.  Centrifugalise   the    milk    sample   for   twenty   to 
thirty  minutes   at   a   speed  of  2500   revolutions   per 
minute. 

3.  Remove  the  motive  power  and  allow  the  machine 
to  slow  down  gradually. 


350 


BACTERIOLOGICAL  ANALYSES. 


4.  Remove  the  tubes  of  milk  from  the  centrifuge. 
Each  tube  will  now  show  (Fig.  170): 

(a)  A  superficial  layer  of  cream  (varying  in  thickness 
with  different  samples)  churned  into  a  semi-solid  mass, 
which  can  be  shown  to  contain  some  organisms  and  a 
few  leucocytes. 

(b)  A  central  layer  of  separated  milk,  thin,  watery, 
and  opalescent,  and  containing  extremely  few  bacteria. 

(c)  A  sediment  or  deposit  consisting  of  the  great 

majority  of  the  contained  bacteria  and 
leucocytes,  together  with  adventitious 
matter,  such  as  dirt,  hair,  epithelial 
cells,  etc. 

•f  .5.  Withdraw  the  rubber  stopper  and 
remove  a  central  plug  of  cream  from 
each  tube  by  means  of  a  sterile  cork 
borer;  place  these  masses  of  cream  in 
sterile  capsules. 

,/  6.  Remove  all  but  the  last  3  or  4  c.c. 
of  separated  milk  from  each  tube,  by 
means  of  sterile  pipettes. 

7.  Mix  the  deposits  thoroughly  with 
the  residual  milk,  pipette  the  mixture 
from  each  tube  into  a  fresh  sterile  tube, 
and  mix  together;  then  fill  with  sterile 
bouillon  (or  normal  saline  solution). 

8.  Place  the  mixed  deposits  in  the 
centrifuge,  counterpoise  with  another  tube  containing 
an  equal  volume  of  water,  and  centrifugalise,  as  before. 

9.  Pipette  off  all  the  supernatant  fluid  and  invert 
the  tube  to  drain  on  to  a  pad  of  sterilised  cotton- wool, 
contained  in  a  beaker.     (This  wool  is  subsequently 
cremated.) 

10.  Examine  both  cream  and  deposit   microscopi- 
cally— 

(a)  In  hanging  drops. 

(b)  In  film  preparations  stained  carbolic  methylene- 


Fig.  170. — Milk  in 
centrifuge  tube. 


EXAMINATION   OF   MILK.  351 

blue,  Gram's  method,  Neisser's  method,  Ziehl-Neelsen's 
method. 

ii.  Adapt  the  final  stages  of  the  investigation  to 
the  special  requirements  of  each  individual  sample, 
as  follows : 

Members  of  the  Typhoid  and  Colon  Groups. — 

Bacillus  Enteritidis  of  Qaertner. — 

Bacillus  Enteritidis  Sporogenes. — 

Vibrio  Cholerse.— 

When  searching  for  any  or  either  of  these  organisms, 
emulsify  the  deposit  with  10  c.c.  sterile  bouillon  and 
proceed  with  the  examination  as  described  under 
water. 

NOTE. — The  B.  coli  communis,  derived  from  the 
alvine  discharges  of  the  cow,  is  almost  universally 
present  in  large  or  small  numbers,  in  retail  milk.  Its 
detection,  therefore,  unless  in  enormous  numbers, 
when  it  indicates  want  of  cleanliness,  is  of  little  value. 

B.  Diphtherias.— 

(A)  i.  Plant  three  sets  of  serial  cultivations  from 
(a)  cream  (twelve  tubes  in  each  set)  upon  oblique 
inspissated  blood-serum,  (6)  deposit  (twelve  tubes  in 
each  set)  upon  oblique  inspissated  blood-serum,  and 
incubate  at  37°  C. 

2.  Pick  off  any  suspicious  colonies  which  may  have 
made   their  appearance  nine  hours  after  incubation, 
and    subcultivate    upon    blood-serum;    return    the 
original  tubes  to  the  incubator. 

3.  Repeat  this  after  eighteen  hours'  incubation. 

4.  From    the    resulting    growths    make    cover-slip 
preparations  and  stain  carbolic  methylene-blue,  Neis- 
ser's method,  Gram's  method. 

5.  Inoculate  guinea-pigs  subcutaneously  with  forty- 
eight-hour-old    glucose    bouillon    cultivation    derived 
from  the  first  subcultivation,  and  observe  the  result. 

6.  Inoculate  guinea-pigs  subcutaneously  with  filtered 


352  BACTERIOLOGICAL   ANALYSES. 

glucose  bouillon  cultivations  (toxins  ?)  and  observe 
the  result. 

(B)  i.  Emulsify  the  remainder  of  the  deposit  with 
3  c.c.  sterile  bouillon  and  inoculate  two  guinea-pigs, 
thus :  guinea-pig  a,  subcutaneously  with  i  c.c.  emulsion; 
guinea-pig  6,  subcutaneously  with  2  c.c.  emulsion;  and 
observe  the  result. 

2.  If  either  or  both  of  the  inoculated  animals  suc- 
cumb, make  complete  post-mortem  examination  and 
endeavour  to  isolate  the  pathogenic  organisms  from 
the  local  lesion. 

Bacillus  Tuberculosis. — Add  5  c.c.  sterile  bouillon 
to  the  deposit  in  the  tube  and  emulsify  thoroughly. 

(A)  i.  Inoculate  each  of  three  guinea-pigs  (previ- 
ously tested  with  tuberculin,  to  prove  their  freedom 
from  spontaneous  tuberculosis)  subcutaneously  at  the 
inner  aspect  of  the  bend  of  the  left  knee,  with  i  c.c. 
of  the  emulsion. 

2.  Introduce  a  small  quantity  of  the  cream  into  a 
subcutaneous  pocket  prepared  at  the  inner  aspect  of 
the  bend  of  the  right  knee  of  each  of  these  three  animals. 
Place  a  sealed  dressing  on  the  wound. 

3.  Observe   carefully,    and   weigh    accurately   each 
day. 

4.  Kill   one  guinea-pig  at  the  end   of  the  second 
week  and  make  a  complete  post-mortem  examination. 
(Compare  Fig.  158,  page  307.) 

5.  If  the  result  of  the  examination  is  negative  or 
inconclusive,  kill  a  second  guinea-pig  at  the  end  of  the 
third  week  and  examine  carefully. 

6.  If  still  negative  or  inconclusive,  kill  the  third 
guinea-pig  at  the  end  of  the  sixth  week.     Make  a  careful 
post-mortem  examination. 

(B)  i.  Place  the  tube  containing  the  remainder  of 
the  emulsion  in  a  water-bath  at  56°  C.  for  ten  minutes. 

2.  Remove  the  tube  from  the  water-bath  and  cool 
rapidly. 


EXAMINATION  OF  MILK.  353 

3.  Inoculate    each    of    two    guinea-pigs,    intraperi- 
toneally,  with  i  c.c.  of  the  emulsion. 

4.  Kill   one  of  the  guinea-pigs  at  the  end  of  the 
first  week  and  examine  carefully. 

5.  Kill  the  second  guinea-pig  at  the  end  of  the  second 
week  and  examine  carefully. 

NOTE. — No  value  whatever  attaches  to  the  result 
of  a  microscopical  examination  for  the  presence  of  the 
B.  tuberculosis  unless  confirmed  by  the  result  of 
inoculation  experiments. 

Streptococcus  Pyogenes  Longus. — 

(A)  i.  Plant  serial  cultivations  from  the  deposit 
upon   (a)  oblique  inspissated  blood-serum   (six  tubes 
in  series)  and  (6)  oblique  nutrient  agar  (six  tubes  in 
series). 

2.  If  the  resulting  growth  shows  colonies  which 
resemble  those  of  the  streptococcus,  make  subcultiva- 
tions  upon  agar  and  in  bouillon  in  the  first  instance 
and  study  carefully. 

(B)  i.  Plant  a  large  loopful  of  the  deposit  into  each 
of  three  tubes  of  glucose  formate  bouillon,  and  incu- 
bate anaerobically   (in  Buchner's  tubes)  for  twenty- 
four  hours  at  37°  C. 

2.  If  the  resulting  growth  resembles   that  of  the 
streptococcus,    make    subcultivations    upon    nutrient 
agar. 

3.  Prepare  subcultivations  of  any  suspicious  colonies 
that  appear,  upon  all  the  ordinary  media,  and  study 
carefully. 

If  the  streptococcus  is  successfully  isolated,  inocu- 
late serum  bouillon  cultivations  into  the  mouse, 
guinea-pig,  and  rabbit,  to  determine  its  pathogenicity 
and  virulence. 

Staphylococcus  Pyogenes  Aureus. — 

i.  Prepare  serial  cultivations  upon  oblique  nutrient 
agar  (eight  tubes  in  series). 
23 


354  BACTERIOLOGICAL   ANALYSES. 

2.  Incubate  at  37°  C.  for  twenty-four  hours. 

3.  Pick  off  any  suspicious  colonies,  plant  on  oblique 
agar,  and  incubate  at  20°  C.     Observe  pigment  forma- 
tion. 

4.  Prepare    subcultivations    from    any    suspicious 
growths  upon  all  the  ordinary  media  and  study  care- 
fully. 

ICE  CREAM. 

Collection  of  the  Sample. — 

1.  Remove  the  sample  from  the  drum  in  the  ladle 
or  spoon  with  which  the  vendor  retails  the  ice  cream, 
and  place  it  at  once  in  a  sterile  copper  capsule,  similar 
to  that  employed  for  earth  samples. 

2.  Pack  for  transmission  in  the  ice-box. 

3.  On  arrival  at  the  laboratory  place  the  copper 
capsules  containing  the  ice  cream  in  the  incubator  at 
20°  C.  for  fifteen  minutes — that  is,  until  at  least  some 
of  the  ice  cream  has  become  liquid. 

Qualitative  and  Quantitative  Examination. — Treat 
the  fluid  ice  cream  as  milk  and  conduct  the  examina- 
tion in  precisely  the  same  manner  as  described  for 
milk  (vide  page  346). 


EXAMINATION  OF  CREAM  AND  BUTTER. 

Collection  of  the  Sample. — Collect,  store,  and  trans- 
mit samples  to  the  laboratory,  precisely  as  is  done  in 
the  case  of  ice  cream. 

Quantitative. — 

Apparatus  Required: 
Sterile  test-tube. 
Sterilised  spatula. 
Water-bath  regulated  at  42°  C. 
Case  of  sterile  plates. 

Case  of  sterile  graduated  pipettes,  i  c.c.  (inhundredths). 
Tubes  of  gelatine-agar  (-J- 10  reaction). 
Plate-levelling  stand,  with  its  water  chamber  filled  with 
water  at  42°  C. 


EXAMINATION  OF  CREAM  AND  BUTTER.        355 

METHOD. — 

1 .  Transfer  a  few  grammes  of  the  sample  to  a  sterile 
test-tube  by  means  of  the  sterilised  spatula. 

2.  Piace  the  tube  in  the  water-bath  at  42°  C.  until 
the  contents  are  liquid. 

3.  Liquefy  eight  tubes  of  gelatine- agar  and  place 
them  in  the  water-bath  at  42°  C.,  and  cool  down  to 
that  temperature. 

4.  Inoculate  the  gelatine-agar  tubes  with  the  fol- 
lowing quantities  of  the  sample  by  the  help  of  a  sterile 
pipette  graduated  to  hundredths  of  a  cubic  centimetre 

—viz.,  o.i,  0.2,  0.3,  0.5,  o.oi,  0.02,  0.03,  and  0.05  c.c. 

5.  Pour  a  plate  cultivation  from  each  of  the  gelatine- 
agar  tubes  and  incubate  at  28°  C. 

6.  "Count"  the  plates  after  three  days'  incubation, 
and  from  the  figures  thus  obtained  estimate  the  number 
of  organisms  present  per  cubic  centimetre  of  the  sample. 

Qualitative. — 

Apparatus  Required: 

Sterile  beaker,  its  mouth  plugged  with  sterile  cotton- wool. 

Scales  and  weights. 

Sterilised  spatula. 

Water-bath  regulated  at  42°  C. 

Separatory  funnel,  250  c.c.  capacity,  its  delivery  tube 
protected  against  contamination  by  passing  it  through 
a  cotton-wool  plug  into  the  interior  of  a  small  Erlen- 
meyer  flask  which  serves  to  support  the  funnel.  This 
piece  of  apparatus  is  sterilised  en  masse  in  the  hot-air 
oven. 

Centrifugal  machine. 

Sterile  tubes  (for  the  centrifuge)  closed  with  solid  rubber 
stoppers. 

Case  of  sterile  pipettes. 

METHOD.— 

1.  Weigh  out  100  grammes  of  the  sample  in  a  sterile 
beaker. 

2.  Plug  the  mouth  of  the  beaker  with  sterile  cotton- 
wool and  immerse  the  beaker  in  a  water-bath  at  42°  C. 
until  the  contents  are  completely  liquefied. 


356  BACTERIOLOGICAL  ANALYSES. 

3.  Fill  the  liquefied  butter  into  the  sterile  separatory 
funnel. 

4.  Transfer  the  funnel  to  the  incubator  at  37°  C. 
and  allow  it  to  remain  there  for  four  days. 

At  the  end  of  this  time  the  contents  of  the  funnel 
will  have  separated  into  two  distinct  strata. 

(a)  A  superficial  oily  layer,   practically  free  from 
bacteria. 

(b)  A  deep  watery  layer,  turbid  and  cloudy  from 
the  growth  of  bacteria. 

5.  Draw  off  the  subnatant  turbid  layer  into  sterile 
centrifugal  tubes,  previously  warmed  to  about  42°  C., 
and  centrifugalise  at  once. 

6.  Pipette  off  the  supernatant  fluid  and  fill  the  tubes 
with  sterile    i   per  cent,   sodium  carbonate    solution 
previously  warmed  slightly;   stopper  the  tubes  and 
shake  vigour ously  for  a  few  minutes. 

7.  Centrifugalise  again. 

8.  Pipette  off  the  supernatant  fluid;  filling  the  tubes 
with   sterile   bouillon,  shake  well,  and  again  centrif- 
ugalise, to  wash  the  deposit. 

9.  Pipette  off  the  supernatant  fluid. 

10.  Prepare  cover-slip  preparations,   fix  and  clear 
as  for  milk  preparations,  stain  carbolic  methylene-blue, 
Gram's  method,  Ziehl-Neelsen's  method,  and  examine 
microscopically  with  a  y^-inch  oil-immersion  lens. 

11.  Proceed  with  the  examination  of  the  deposit 
as  in  the  case  of  milk. 


EXAMINATION  OF  UNSOUND  MEATS. 

(INCLUDING  TINNED  OR  POTTED  MEATS,  FISH,  ETC.) 

Qualitative. — 

Apparatus  Required: 

Erlenmeyer  flask  (500  c.c.  capacity)  containing  250  c.c. 

sterile  bouillon  and  fitted  with  solid  rubber  stopper. 
Scissors  and  forceps. 


EXAMINATION  OF   UNSOUND   MEATS.  357 

Water  steriliser. 

Hypodermic  syringe. 

Case  of  sterile  capsules. 

Filtering  apparatus  as  for  water  analysis. 

Case  of  sterile  plates. 

Case  of  sterile  graduated  pipettes,  10  c.c.  (in  tenths  of  a 

cubic  centimetre). 
Case  of  sterile  graduated  pipettes,   i  c.c.  (in  tenths  of  a 

cubic  centimetre). 
Plate-levelling  stand. 
Tubes  of  nutrient  gelatine. 
Tubes  of  nutrient  agar. 
Water-bath  regulated  at  42°  C. 
Bulloch's  apparatus. 

METHOD.— 

(A)  i.  Mince  a  portion  of  the  sample  by  the  aid  of 
sterile  scissors  and  forceps,  and  add  the  minced  sample 
to  the  bouillon  in  the  flask. 

2.  Make  an  extract  by  standing  the  flask  in  the 
water-bath  at  42°  C.   for  half  an  hour,  shaking  its 
contents  from  time  to  time. 

3.  Pipette  off  10  c.c.  of  the  extract  into  a  sterile 
test-tube  and  remove  for  use  under  section  C. 

4.  Filter    the    extract    through    a    sterile    Berkfeld 
filter. 

5.  Emulsify  the  bacterial  residue  with  10  c.c.  sterile 
bouillon. 

6.  Pour  a  set  of  gelatine  and  a  set  of  agar  plates  from 
tubes  containing  0.2,  0.3,  and  0.5  c.c.  of  the  extract, 
and  incubate  aerobically,  the  gelatine  set  at  20°  C., 
the  agar  at  37°  C. 

7.  Pour  duplicate  sets  of  gelatine  and  agar  plates 
and  incubate   anaerobically  in   Bulloch's   apparatus, 
at  similar  temperatures. 

8.  Subcultivate  from  the  colonies  that  make  their 
appearance  and  identify  the  various  organisms. 

9.  Continue  the  investigations  with  reference  to  the 
detection  of  pathogenic  organisms  as  described  under 
water  (page  327  et  seq.). 


BACTERIOLOGICAL  ANALYSES. 

(B)  i.  Feed  rats  and  mice  on  portions  of  the  sample 
and  observe  the  result. 

2.  If  any  of  the  animals  die,  make  complete  post- 
mortem examinations  and  endeavour  to  isolate  the 
pathogenic  organisms. 

(C)  i.  Inoculate  rats,   mice,  and  guinea-pigs  sub- 
cutaneously  and  intraperitoneally  with  various  quan- 
tities of  the  bouillon  extract,  and  observe  the  result. 

2.  If  any  of  the  animals  succumb  to  the  inocula- 
tion, make  careful  post-mortem  examinations  and  en- 
deavour to  isolate  the  pathogenic  organisms. 

EXAMINATION   OF   FILTERS. 

Porcelain  filter  candles  are  examined  with  reference 
to  their  power  of  holding  back  all  the  micro-organisms 
suspended  in  the  fluids  which  are  filtered  through 
them,  and  permitting  the  passage  of  only  germ-free 
filtrates.  The  examination  is  conducted  as  follows: 

Apparatus  Required: 

Filtering  apparatus — one  or  other  of  those  described  under 
Examination  of  Water.  The  actual  filter  candle  that  is 
used  must  be  the  one  it  is  intended  to  test ;  the  arrange- 
ment of  the  apparatus  will  therefore  need  to  be  varied 
with  each  different  form  of  filter  (see  also  pages  47,  48). 

Plate-levelling  stand. 

Case  of  sterile  plates. 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths). 

Case  of  sterile  pipettes,  i  c.c.  (in  tenths). 

Tubes  of  nutrient  gelatine. 

Flask  containing  sterile  normal  saline  solution. 

Sterile  measuring  flask,  1000  c.c.  capacity. 

METHOD. — 

1.  Prepare  surface  cultivations,  on  nutrient  agar  in 
a  culture  bottle,  of  the  Bacillus  prodigiosus,  and  incu- 
bate at  20°  C.,  for  forty-eight  hours. 

2.  Pipette  5  c.c.  sterile  normal  saline  into  the  culture 
bottle  and  emulsify  the  entire  surface  growth  in  it. 

3.  Pipette  the  emulsion  into  the  sterile  measuring 


EXAMINATION   OF  DISINFECTANTS.  359 

flask  and  dilute  up  to   1000  c.c.  by  the  addition  of 
sterile  water. 

4.  Pour  the  emulsion  into  the  filter  reservoir  and 
start  the  filtration. 

5.  When  the  filtration  is  completed,  pour  six  gela- 
tine plates  each  containing  i  c.c.  of  the  filtrate. 

6.  Incubate  at  20°  C.  until,  if  necessary,  the  comple- 
tion of  seven  days. 


EXAMINATION  OF   DISINFECTANTS. 

Disinfectants  or  Germicides  are  examined  with 
reference  to  three  points: 

(A)  Inhibition  coefficient — i.  e.,that  percentage  of 
the    disinfectant    present    in    the    nutrient    medium 
which  is  sufficient  to  prevent  the  growth  and  multi- 
plication of  bacteria  therein. 

(B)  Inferior  lethal  coefficient — i.  e.,  the   time  ex- 
posure necessary  to  kill  vegetative  forms  suspended 
in  water  at  20°  to  25°  C.,  in  which  the  disinfectant  is 
present  in  medium  concentration    (concentration  in- 
sufficient to  cause  plasmolysis). 

(C)  Superior  lethal  coefficient — i.  e.y  the  time   ex- 
posure necessary  to  kill  spores  under  conditions  similar 
to  those  obtaining  in  B. 

The  methods  here  detailed  only  specifically  refer 
to  those  disinfectants  mentioned  under  Germicides  in 
the  Scheme  for  the  Study  of  Bacteria  (page  252),  but 
the  technique  is  practically  similar  for  all  other  chemi- 
cal disinfectants. 

Inhibition  Coefficient. — 

Apparatus  Required: 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths). 

Case  of  sterile  pipettes,  i  c.c.  (in  tenths). 

Sterile  tubes  or  capsules  for  dilutions. 

Tubes  of  nutrient  bouillon. 
Materials  Required: 

i .  Five  per  cent,  aqueous  solution  of  carbolic  acid. 


360  BACTERIOLOGICAL   ANALYSES. 

2.  One  per  cent,  aqueous  solution  of  perchloride  of  mer- 

cury. 

3.  One-tenth  per  cent,  aqueous  solution  of  formaldehyde. 

METHOD.— 

1.  Prepare    a    series    of   six    tube    cultivations,    in 
bouillon   (each  tube  containing   10  c.c.   of  medium), 
of  each  organism  employed  in  the  test  and  add  2  c.c. 
of  the  5  per  cent,  carbolic  acid  solution  (i  :  100)  to 
the    first,    i   c.c.    (i  :  200)    to    the   second,    0.6  c.c. 
(i  :  300)  to  the  third,  0.5  c.c.  (i  :  400),  to  the  fourth, 
0.4  c.c.  (i  :  500)  to  the  fifth,  and  0.2  c.c.  (i  :  1000)  to 
the  sixth. 

2.  Prepare  a  similar  series  of  tube  cultivations  and 
add  o.i  c.c.   (i  :  1000),  0.05  c.c.    (i  :  2000),  0.03  c.c. 
(i  :  3000),  0.025  c.c.  (i  :  4500),  0.02  c.c.  (i  :  5000),  and 
o.oi  c.c.  (i  :  10,000)  of  the  i  per  cent,  perchloride  of 
mercury  solution. 

3.  Prepare  a  similar  series  of  tube  cultivations  and 
add  i  c.c.  (i :  1000),  0.4  c.c.  (i :  2500),  0.2  c.c.  (i :  5000), 
o.i  c.c.    (i  :  10,000),  0.075  c.c.  (i  :  15,000),  and  0.05 
c.c.    (i  :  20,000)  of  the    o.i   per  cent,  formaldehyde 
solution. 

4.  Incubate  all  three  sets  of  cultivations  under  opti- 
mum conditions  as  to  temperature  and  atmosphere. 

5.  Examine  each  of  the  culture  tubes  from  day  to 
day,  until   the  completion  of   seven   days,  and  note 
those  tubes,  if  any,  in  which  growth  takes  place. 

Inferior  Lethal  Coefficient. — 

Apparatus  Required: 

Highly  concentrated  solutions  of  the  disinfectants. 

Sterile  test-tubes  in  which  to  make  dilutions  from  the  con- 
centrated solutions  of  the  disinfectants. 

Hanging-drop  slides. 

Cover-slips. 

Krlenmeyer  flask  containing  100  c.c.  sterile  distilled  water. 

Case  of  sterile  pipettes,  10  c.c.  (in  tenths  of  a  cubic  centi- 
metre). 

Case  of  sterile  pipettes,  i  c.c.  (in  tenths  of  a  cubic  centi- 
metre). 


EXAMINATION   OF   DISINFECTANTS.  361 

METHOD.— 

1.  Prepare   a  surface  cultivation    of  each    of    the 
"test"   organisms   upon  nutrient  agar  in   a  culture 
bottle   and   incubate   under   optimum   conditions   for 
forty-eight  hours;  then  examine  the  cultivation  micro- 
scopically to  determine  the  absence  of  spores. 

2.  Prepare  solutions  of  different  percentages  of  each 
disinfectant. 

3.  Make  a  series  of  hanging-drop  preparations  from 
the  agar  culture,  using  a  loopful  of  disinfectant  solu- 
tion of  the  different  percentages  to  prepare  the  emulsion 
on  each  cover-slip. 

4.  Examine  microscopically  and  note  the  strongest 
solution  which  does  not  cause  plasmolysis   and  the 
weakest  solution  which  does  plasmolyse  the  organism. 

5.  Make  control  preparations  of  these  two  solutions 
and  determine  the  percentage  to  be  tested. . 

6.  Pipette    10   c.c.    sterile   water   into    the    culture 
bottle  and  suspend  the  entire  surface  growth  in  it. 

7.  Transfer  the  suspension  to  the  Erlenmeyer  flask 
and  mix  it  with  the  90  c.c.  of  sterile  water  remaining 
in  the  flask. 

8.  Pipette  10  c.c.  of  the  diluted  suspension  into  each 
of  ten  sterile  test-tubes. 

9.  Label  one  of  the  tubes  "Control"  and  place  it 
in  the  incubator  at  20°  C. 

10.  Add  to  each  of  the  remaining  tubes  a  sufficient 
quantity  of  a  concentrated  solution  of  the  disinfectant 
to  produce  the  percentage  previously  determined  upon 
(vide  step  5). 

11.  Incubate  the  tubes  at  20°  C. 

12.  At   hourly   intervals   remove   the   control   tube 
and  one  of  the  tubes  with  added  disinfectant  from  the 
incubator. 

13.  Make  a  subcultivation  from  both  the  control  and 
the  test  suspension,  upon  the  surface  of  nutrient  agar; 
incubate  under  optimum  conditions. 


362         BACTERIOLOGICAL,  ANALYSES. 

14.  Observe  these  culture  tubes  from  day  to  day 
until  the  completion  of  seven  days,  and  determine 
the  shortest  exposure  necessary  to  cause  the  death  of 
vegetative  forms. 

Superior  Lethal  Coefficient. — 

1.  Prepare  surface  cultivations  of  the  "  test "  organ- 
isms upon  nutrient  agar  in  a  culture  bottle,  and  incu- 
bate under  optimum  conditions,  previously  determined, 
for  the  formation  of  their  spores. 

2.  Employ  that  percentage  solution  of  the  disinfec- 
tant determined  in  the  previous  experiment,  and  com- 
plete the  investigations  as  detailed  therein,  steps  6  to 
14,  increasing  the  interval  between  planting  the  sub- 
cultivations  to  two,  three,  or  five  hours  if  considered 
advisable. 

NOTE. — Where  it  is  necessary  to  leave  the  organisms 
in  contact  with  a  strong  solution  of  the  disinfectant 
for  lengthy  periods,  some  means  must  be  adopted  to 
remove  every  trace  of  the  disinfectant  from  the  bac- 
teria before  transferring  them  to  fresh  culture  media; 
otherwise,  although  not  actually  killed,  the  presence 
of  the  disinfectant  may  prevent  their  development, 
and  so  give  rise  to  an  erroneous  conclusion.  In  such 
cases  proceed  as  follows : 

1.  Transfer   the   suspension   of   bacteria   to    sterile 
centrifugal  tubes;  add  the  required  amount  of  dis- 
infectant, and  allow  it  to  remain  in  contact  with  the 
bacteria  for  the  necessary  period. 

2.  Centrifugalise  thoroughly,  pipette  off  the  super- 
natant fluid;  fill  the  tube  with  sterile  water  and  dis- 
tribute the  deposit  evenly  throughout  the  fluid. 

3.  Centrifugalise  again,  pipette  off  the  supernatant 
fluid;  fill  the  tube  with  sterile  water;  distribute  the 
deposit  evenly  throughout  the  fluid,  and  transfer  the 
suspension  to  a  litre  flask. 

4.  Make  up  to  a  litre  by  the  addition  of  sterile 
water;  filter  the  suspension  through  a  sterile  porcelain 
candle. 

5.  Emulsify  the  bacterial  residue  with  5  c.c.  sterile 
bouillon. 

6.  Prepare  the  necessary  subcultivations  from  this 
emulsion. 


INDEX. 


ABBA'S  condenser,  56 
Aberration,  chromatic,  57 

spherical,  56 
Absolute  alcohol  as  a  fixative,  75 

as  an  antiseptic,  33 
Absorbent  paper  for  drying  cover- 
slips,  65 

A.  C.  E.  mixture,  266 
Acetic  acid  for  clearing  films,  76 
Acid-fast  bacilli,  to  stain,  95,  106 
Acid   production,   analysis  table, 

227,  228 
by  bacteria,  224 
qualitative  examination,  225 
quantitative        examination, 

225 

Actinomyces  bo  vis,  312 
Action  of  different  gases  on  bac- 
teria, 240 

Aerobic  cultures,  177 
Aerogenic  bacteria,  111 
Agar  expansion  table,  135 
gelatine,  153 

method  of  preparation,  149 
rapid  method  of  preparing,  150 
Agglutination  reaction,  252 

macroscopical  observation  of, 

259 
microscopical  observation  of, 

256 
Air,  analysis  of,  qualitative,  338 

quantitative,  335 
filter,  43 
pump,  45 

Albumin  solution,  Mayer's,  103 
Alkaline  serum  agar,  157 
Ammonia  production,  229 
Amphitrichous  bacteria,  115 
Anaerobic  cultures,  186 
Anaesthetics,  266 
Analysis  of  air,  apparatus  for,  335 
method  of,  336 
qualitative       bacteriological, 

338 
quantitative    bacteriological, 

335 

of  butter,  qualitative  bacterio- 
logical, 355 


Analysis  of  butter,  quantitative 

bacteriological,  354 
of  cream,  qualitative  bacterio- 
logical, 355 
quantitative    bacteriological, 

354 

of  ice-cream,    qualitative   bac- 
teriological, 354 
of  meat,  apparatus  for,  356 
method  of,  357 
qualitative       bacteriological, 

375 

of  milk,  apparatus  for,  345 
collection  of  samples,  344 
method  of,  346 
qualitative       bacteriological, 

347 
quantitative    bacteriological 

344 

of  sewage,  qualitative  bacterio- 
logical, 335 
quantitative    bacteriological, 

334 

of  soil,  apparatus  for,  339 
collection  of  samples,  338 
method  of,  340 
qualitative       bacteriological, 

342 
quantitative    bacteriological, 

339 

of  water,  apparatus  for,  324 
method  of,  326 
qualitative       bacteriological, 

323 
quantitative    bacteriological, 

316 

Anatomy  of  bacteria,  113 
Aniline  dyes,  77 

gentian  violet,  85 
Anthrax,  bacillus  of,  305 
Antiseptics,  33 
action  of,  359 

Arnold's  steam  sterilizer,  39 
Ascitic  bouillon,  156 
Ascomycetae,  109 
Ascospores,  110 
Asparagin  media,  171 
Aspergillus,  108 


363 


364 


INDEX. 


Atmospheric  conditions,  239 

Attenuating  the  virulence  of  or- 
ganisms, 285 

Autoclave,  42 

Autopsy,  method  of  conducting, 
287 

BACILLUS,  112 
aegyptiacus,  313 
anthracis,  305 

in  water,  333 
aquatilis  sulcatus,  304 
botulinus,  309 
chauvei,  309 
coli  communis,  304 

in  water,  327 
diphtherias,  302 

in  milk,  351 
enteritidis  of  Gartner,  304 

in  water,  327 
sporogenes,  309 
in  water,  331 
fluorescens  liquefaciens,  300 

non-liquefaciens,  300 
influenzas,  313 
leprae,  308 
mallei,  314 
mycoides,  305 
oedematis  maligni,  309 

in  soil,  343 

of  avian  tubercle,  306 
of  Friedlander,  299 
of  Hoffmann,  302 
of  rhinoscleroma,  299 
of  symptomatic  anthrax,  309 
pestis,  310 
phlei,  306 
pyocyaneus,  300 
septicaemiae  haemorrhagicae,  310 
subtilis,  305 
suipestifer,  310 
tetani,  309 

in  water,  333 
tuberculosis,  306 

in  milk,  352 
typhi  abdominalis,  304 

in  water,  327 
typhosus,  304 
xerosis,  302 

Bacteria,  classification  of,  111 
microscopical    examination    of, 

stained,  80 
unstained,  72 
Bacterial  enzymes,  222 

food-stuffs,  121 
Base  of  microscope,  52 
Beer  wort,  165 
Beet-root  medium,  164 
Beggiotoa,  1 1 3 


Benzole  bath,  202 
Berkfeld  filter,  45 
Bile-salt  agar,  169 

broth,    169 

Biochemistry  of  bacteria,  221 
Bismarck  brown,  84 
Blastomycetes,     morphology     of, 

109 

Blood  agar,  158 
pipettes,  22 

serum,  collection  of,  153 
inspissated,  155,  156 
to  inspissate,  155 
Body  tube  of  microscope,  52 
Botkin's  anaerobic  method,  192 
Bouillon,  preparation  of,  141 
Brain  agar,  150 
Bread  paste,  167 
Brownian  movement,  74 
Buchner's  anaerobic  method,  189 
Bulloch's  anaerobic  method,   194 

tubes,  292 
Butter,  analysis  of,  apparatus  for, 

355 

method  of,  355 
qualitative,  355 
quantitative,  354 

CAGES,  263 

for  guinea-pigs,  264 

for  mice,  263 

for  rabbits,  264 

for  rats,  264 

Camera  lucida,  Abbe,  60 
Capillary  pipettes,  20 

graduated,  23 
Capsule  of  bacteria,  113 

thermo-regulator,    175 

to  stain  the,  86,  105 
Capsules,  collodion,  inoculation  of, 

279 
preparation  of,  279 

glass,  20 

to  clean  infected,  26 

to  clean  new,  25 

to  sterilise,  36 
Carbolic  acid,  33 
Carbolised  agar,  152 

bouillon,  144 

gelatine,  149 
Carbon  dioxide,  233 
Carrot  medium,  164 
Cell  wall  of  bacteria,  114 
Centrifugalised  milk,  350 
Centrifugal  machine,  349 
Chemical  products  of  bacteria,  221 
Chloroform,    33 
Cholera,  302 
Chromatic  aberration,  57 


INDEX. 


365 


Chromogenic  bacteria,  111 
Cladothrix,  113 

nivea,  312 

Classification  of  bacteria,  111 
Clearing  films  with  acetic  acid,  76 
Coarse  adjustment,  53 
Cocaine,  266 
Coccus,  110 
Coefficient,  inferior  lethal,  360 

of  inhibition,  359 

superior  lethal,  359 
Cohn's  solution,  171 
Collection  of  pus,  271 

of  water  samples,  316 
Collodion  capsules,  279 
Coloured  light,  action  of,  252 
Columella,  108 
Compensation  eyepiece,  56 
Conidia,  109 

Continuous  sterilisation,  41 
Corrosive  sublimate,  Lang,  76 
Cotton-wool  filter,  43 
Counterstaining,  78 
Cover-slip  films,  75 
Cover-slips,  27 

to  clean  new,  28 

used,  28 

Crates  for  test-tubes,  36 
Cream,  analysis  of,  apparatus  for, 

355 

method  of,  355 
qualitative,  355 
quantitative,  354 
Crenothrix,  113 
Culture  bottles,  19 

flasks,  19 
Cutaneous  inoculation,  274 

DAUGHTER  cells,  110 
Daylight,  diffuse,  action  of,  250 
Decolourising  agents,  78 
Definition  of  objective,  57 
Description  of  plate  culture,  207 
Descriptive  terms,  208 
Desiccation,  effect  of,  249 
Desiccator,  Miiller's,  249 
Diaphragm,  iris,  55 
Diastatic  enzymes,  223 
Differential  steriliser,  202 
Diluting  chamber,  196 
Diphtheria,  bacillus  of,  302 
Diplobacillus,  112 
Diplococcus,  111 

pneumonise,  292 
Discontinuous  sterilisation,  40 
Discs  of  plaster-of -Paris,  173 
Disinfectants,  33 

action  of,  359 

testing  power  of,  359 


Dosage  of  inoculum,  269 
Double  nosepiece,  58 
Dropping  bottles,  68 
Dry  heat,  34 
Dunham's  solution,  168 
Dyes,  aniline,  77 

EDGE  of  individual  colonies,  char- 
acters of,  213 
Egg-albumen  media,  160 
Egg  to  clear  nutrient  media  with, 

145 

Eisenberg's  milk-rice  medium,  167 
Elevation  of  colonies,  description 

of,  209 

Eisner's  gelatine,  164 
Endogenous  spores,  varieties  of, 

118 

English  proof  agar,  1 70 
Enumerating  discs,  Jeffer's,  322 

Fakes',  322 
Enumeration  of  micro-organisms, 

321 
Environmental    conditions,     121, 

238 
Enzyme  production  by  bacteria, 

222 

Eosin,  83 

Esmarch's     anaerobic     culture 
method,  187 

roll  culture,  199 

water-bottle,  317 
Estimation  of  reaction  of  media, 

128 

Ether,  33 
Eucaine,  266 
Exalting  virulence  of  organisms, 

284 
Expansion  table  for  agar,  135 

for  gelatine,  134 
Experimental  animals,  261 

inoculation  of  animals,  261 
Extracellular   toxins,    testing  of/ 

261 

Eyepieces,  56 
Eye-screen,  59 

FEEDING  experiments,  284 

Fermentation  tubes,  24 

Field  of  objective,  57 

Filar  micrometer,  63 

Filling  tubes,  etc.,  with  medium, 

.     138 

Film  preparations,  fixing,  75 

making,  75 

staining,  76 

Filter   candles,   testing  efficiency 
of,  358 

to  disinfect,  34 


366 


INDEX. 


Filter  candles,  to  sterilise,  34 

flasks,  19 

papers,  136 

to  fold,  136 
Filtering  agar,  137 

gelatine,  137 
Filters,  43 

Berkfeld,  45 

Chamberland,  44 

cotton-wool,  43 
Filtration  by  aspiration,  45 

of  media,  137 

under  pressure,  47 
Fine  adjustment,  54 
Fish,   bacteriological  analysis  of, 
356 

bouillon,   162 

gelatine,  162 

agar,  163 

Fission,  multiplication  by,  116 
Fixation  by  heat,  75 

of  tissues,  98 
Fixing  fluids,  75 
Flagella,  115 

to  stain,  87 
Flasks,  Bohemian,  18 

Erlenmeyer's,  18 

filter,  19 

Kolle's  culture,  19 

to  clean  infected,  28 

to  clean  new,  25 

to  plug,  29 

to  sterilise,  36 

Fluid  media,  description  of,  217 
Foot  of  microscope,  52 
Formaldehyde,  33 
Formalin    method    of    preserving 

cultures,  291 
tissues,  292 

Fractional  sterilisation,  37,  38 
Frankel  and  Voges'  solution,  171 
Freezing  method  of  sectioning,  98 
French  proof  agar,  170 
Fresh  preparations  of  bacteria,  69 
Fuchsin,  82 

GAS  analysis,  qualitative,  235 

quantitative,  235 
collecting  apparatus,  236 
production  by  bacteria,  233 
tubes  for  media,  139 

Gasperini's  solution,  167 

Gelatine  agar,  153 
expansion  table,  134 
method  of  preparation,  145 
rapid  method    of    preparation. 
145 

General  anaesthetics,  266 

Gentian  violet,  83 


German  lined  paper,  65 
Germicides,  33 

testing  power  of,  359 
Geryk  air-pump,  45 
Glanders,  bacillus  of,  314 
Glucose  formate  agar,  152 
bouillon,  142 
gelatine,  148 
Glycerinated  potato,  164 
Glycerine  agar,  151 

blood-serum,  155 

bouillon,  142 

potato  broth,  164 
Gpadby's  gelatine,  165 
Gonidium,  108 
Goniodophore,  109 
Gonococcus,  297 
Graduated  pipettes,  20 
Gram's      differential      staining 

method,  93 
Gram-Weigert    staining    method, 

94,  104 

Grease  pencils,  68 
Griiber's  reaction,  252 
Guinea-pig  cages,  264 
Gulland's  solution,  76 
Gum  solution,  99 

H^MATOCYTOMETER  Cell,    196 

Haematoxylin,  85 
Hanging-drop  cultures,  184 

preparation,  72 
examination  of,  72 
staining  of,  74 

slides,  65 

Hardening  tissues,  98 
Hay  infusion,  165 
Hearson's  water-bath,  242 
Heat,  effect  of,  242 
Heiman's  serum  agar,  157 
Hesse's  anaerobic  culture  method, 

187 

Hog  cholera  bacillus',  310 
Holder  for  guinea-pigs,  273 

for  mice,  274 
Hot  air,  35 
Hot-air  oven,  35 

to  use  the,  36 
Hot-water  funnel,  138 
Human  blood  agar  plates,  198 
Huyghenian  eyepiece,  56 
Hydrogen,  detection  of,  233 

generating  apparatus,  191 
Hypha,  107 
Hyphomycetes,  107 

morphology  of,  107 

reproduction  of,  107 

ICE-BOX  for  water  samples,  317 


INDEX. 


367 


Ice-cream,  analysis  of,  qualitative, 

354 

quantitative,   354 
Impression  films,  79 
Incubators,  174 
Indol  production,  230 
Inferior  lethal  coefficient,  360 
Influence  of  environment  on  bac- 
terial growth,  121 
Inhalation  experiments,  283 
Inhibition  coefficient,  359 
Inoculating  syringe,  265 
Inoculation,  cutaneous,  274 

intracranial,  280 

intramuscular,  276 

intraocular,  280 

intraperitoneal,  277 

intrapulmonary,  281 

intravenous,  281 

of  bacteria,  effects  of,  291 

of  collodion  capsules,  279 

subcutaneous,  275 
Inoculum,  character  of,  267 

preparation  of,  267 
Inosite-free  bouillon,  141 
Insoluble  toxins,  testing  of,  260 
Intermittent  sterilisation,  40 
Intracellular    toxins,    testing    of, 

260 

Intracranial  inoculation,  280 
Intramuscular  inoculation,  276 
Intraocular  inoculation,  280 
Intraperitoneal  inoculation,  277 
Intrapulmonary  inoculation,   281 
Intravenous  inoculation,  281 
Invertin  enzymes,  223 
Involution  forms,  116 
Iodine  solution,  94 
Iron  bouillon,  143 

peptone  solution,  168 
Isolation  by  animal  experiment, 
204 

by  differential  atmosphere,  203 
incubation,  201 
media,  200 
sterilisation,  201 

by  dilution,  196 

by  plate  cultures,  197 

of  sporing  bacteria,  201 

JEFFER'S  enumerating  discs,  322 

KAISERLING  solution,  292 
Kanthack's  serum  agar,  157 
Killed  cultivations,  260 
Kipp's  hydrogen  apparatus,  191 
Kitasato's  serum  flasks,  19 
Klebs-Loffler  bacillus,  302 
Koch's  steam  steriliser,  39 


Koch-Week's  bacillus,  313 
Kolle's  culture  flasks,  19 

LAB.  enzymes,  224 
Lactose  litmus  agar,  152 

bouillon,  141 

gelatine,  148 
Lakmus  molke,  144 
Lang's  solution,  76 
Lead  bouillon,  143 
Leptothrix,  112,  113 
Lethal  dose,  minimal,  269 
Light,  action  of,  250 
Liquid  soap,  267 
Lithium  carmine,  84 
Litmus  bouillon,  141 
gelatine,  148 
milk,  161 

cultivations,   descriptions  of, 

217 

whey,  161 

Local  anaesthetics,  266 
Loffier's  serum,  156 
Lophotrichous  bacteria,  116 
Lorrain  Smith's  serum,  156 
Lugol's  solution,  94 
Lysol,  33 

MAcCoNKEv's  capsule  stain,  86 

media,  169 

MacCrorrie's  flagella  stain,  89 
Macroscopical  examination  of  cul- 
tures, 207 
Malta  fever,  315 

Margin  of  individual  colonies,  213 
Material  for  inoculation,  267 
Mayer's  albumin,  103 
Measuring  bacteria,  60 
Meat,  bacteriological  analysis  of, 

356 
extract,  127 

reaction  of,  128 
Mechanical  stage,  54 

tube  length,  53 
Media,  culture: 

agar  gelatine  (Guarnieri),  152 

ascitic  bouillon,  156 

asparagin  medium  (Frankel  and 

Voges),  171 
(Uschinsky),  171 
beer  wort,  165 
beet-root,  164 
bile-salt  agar  (MacConkey),  169 

broth  (MacConkey),  169 
blood-agar  (Washbourn),  158 
blood-serum,  153 
(Loffler),  156 
(Lorrain  Smith),  156 
brain  agar,  150 


368 


INDEX. 


Media,  culture: 
bread  paste,  167 
carbolised  agar,  152 
carrot,  164 
Cohn's  solution,  171 
egg-albumen,  160 

(Tarchanoff  and  Kolesnikoff), 

160 

English  proof  agar  (Blaxall) ,  1 70 
fish  bouillon,  162 
gelatine,    162 

agar,  163 
French  proof  agar  (Sabouraud), 

170 

gelatine  agar,  152 
glucose  formate  agar,  152 
bouillon  (Kitasato),  142 
gelatine  (Kitasato),  148 
glycerinated  potato,  164 

broth,  164 
glycerine  agar,  151 
blood -serum,  155 
bouillon,  142 
hay  infusion,  165 
inosite-free      media      bouillon 

(Durham),  141 
iron  bouillon,  143 

peptone  solution  (Pakes),  168 
lactose  litmus  agar  (Wurtz),  152 
bouillon,  144 
gelatine  (Wurtz),  148 
Lakmus  molke,  144 
lead  bouillon,  143 
litmus  bouillon,  143 
gelatine,  148 
milk,  161 
whey,  161 
milk,  160 

rice  (Eisenberg),  167 

(Soyka),   167 
Naegeli's  solution,  171 
nitrate  bouillon,  143 

water  (Pakes),  168 
nutrient  agar-agar,  149 
bouillon,  141 
gelatine,  145 

rapid  method  of  preparing, 

145 

parsnip,  164 
Pasteur's  solution,  170 
peptone  water  (Dunham),  168 

rosolic  acid  water,  168 
plaster-of -Paris  discs,  173 
potato,  163 

gelatine  (Eisner),  164 

(Goadby),  165 
serum  agar  (Heiman),  157 

(Kanthack    and    Stevens), 
157 


Media,  culture: 

serum  agar  (Wertheimer),  156 

bouillon,  156 

silicate  jelly  (Winogradsky),  172 
spleen  agar,  150 
sugar  agar,  151 
bouillon,  142 
gelatine,  148 

sulphindigotate  agar,  152 
bouillon  (Weyl),  143 
gelatine  (Weyl),  148 
turnip,  164 
urine  agar,  159 
gelatine,  158 

(Heller),  159 

wheat  broth  (Gasperini),  167 
whey  agar,  162 
gelatine,  161 
wine  must,  167 

Winogradsky's     solution     (for 
nitric  organisms),  172 
(for  nitrous  organisms), 

172 

wort  agar,  166 
gelatine,  166 

yeast  water  (Pasteur),  170 
Media,  filtration  of,  137 
Media,  tubing,  138 
Medium  store  boxes,  140 
Merismopedium,  111 
Mesophilic  bacteria,  122 

pathogenic  effects,  260 
Metabolic    products    of   bacteria, 

123 

Metachromatic  granules,  1 1 5 
Metal  instruments,  to  sterilise,  37, 

38 

Methods  of  identification  of  bac- 
teria, 205 
of  inoculation,  274 
Methylene-blue,  81 
Meyer's  carmine,  85 
Micrococcus,  110 
agilis,  296 
candicans,  296 
melitensis,  315 
tetragenus,  297 
Micrometer,  59 
filar,  63 
net,  62 
ocular,  61 
stage,  60 

Micrometry,  methods  of,  60 
Micron,  60 

Microscope  for  bacteriology,  51 
Microscopical  examination  of  bac- 
teria, 218 
stained,  80 
unstained,  72 


INDEX. 


369 


Milieu  d'epreuve,  170 
Milk,  analysis  of,  qualitative,  347 
quantitative,   345 

medium,  160 

rice  media,  167 
Minimal  lethal  dose,  269 
Mirrors  for  microscope,  56 
Moist  heat,  37 
Molecular  movement,  74 
Monotrichous  bacteria,  115 
Motility,  examination  for,  72 
Moulds,  examination  of,  107 

for  paraffin  imbedding,  102 
Mounting  film  preparations,  78 

paraffin  sections,  102 
Mouse  cages,  263 

holder,  274 

scales,  263 
Mucorinae,  107 
Mucor  mucedo,  108 
Muffle  furnace,  34 
Miiller's  desiccator,  249 
Museum    specimens,    preparation 

of  cultures  for,  291 
of  tissues  for,  292 
Mycelium,  107 
Mycoprotein,  115 

NAEGEU'S  solution,  171 
Naked  flame,  34 
Neisser's  staining  method,  95 
Net  micrometer,  62 
Nitrate  bouillon,  143 

water,    169 

Nitric  organisms  in  soil,  343 
Nitroso-indol  reaction,  230 
Nitrous  organisms  in  soil,  343 
Nosepiece,  double,  58 

triple,  58 
Novy's  anaerobic  method,  193 

jars,  193 

Numerical  aperture,  57 
Nutrient  media,  125 

OBJECTIVES,  56 
Ocular  micrometer,  61 
Oculars,  56 
Oese,  .66 
Oidium,  109 
Oil  of  garlic,  33 

of  mustard,  33 

Operating  table  for  animals,  272 
Optical  characters  of  colonies  213 

tube  length,  53 

Optimum  reaction  of  medium,  de- 
termination of,  248 

temperature,  determination  of, 
241 


24 


Orsat-Lunge  gas  analysis  appara- 
tus, 235 
Orth's  carmine,  84 

FAKES'  counting  disc,  322 

filter  reservoir,  49 
Papier  Chardin,  137 
Paraffin  sections,  mounting,  102 

staining,  103 
Parietti's  bouillon,  144 
Parsnip  medium,  164 
Passages  of  virus,  285 
Pasteur-Chamberland  filter,  44 
Pasteur  pipettes,  21 

solutions,  170 
Pathogenesis,  methods  of  testing, 

259 

Pathogenic  bacteria,  111 
Pediococcus,  111 
Penicillium,  108 
Peptone  rosolic  acid  water,  168 

water,    168 

Perchloride  of  mercury,  33 
Perisporiaceae,  108 
Peritrichous  bacteria,  1 16 
Permanent   preparations  of   bac- 
teria, 291 
of  tissues,  292 
Petri's  dishes,  19 
Phenol  production,  231 
Photogenic  bacteria,  111 
Picric  acid  solution,  104 
Picrocarmine,  84 
Pigment  production,  232 
Pipettes,  20,  21,  22,  23 

to  clean  infected,  27 
new,  25 

to  sterilise,  36 
Plasmolysis,  114 
Plaster-of -Paris  discs,  173 
Plate  box,  20 

cultures,    181 

levelling  stand,  182 
Plates,  19 

to  clean  infected,  26 
new,  25 

to  sterilise,  36 
Platinum  needles,  66 

method  of  mounting,  67 
Pneumobacillus,  299 
Pneumococcus,  298 
Polar  granules,  115 
Porcelain  filter,  44 
Post-mortem  examinations,  287 
Potato  gelatine,  164,  165 

medium,   163 

Potted  meat,  bacteriological  anal- 
ysis of,  356 
Pouring  plates,  182,  183 


370 


INDEX. 


Primary  colours,  action  of,  252 
Proteolytic  enzymes,  222 
Psychrophilic  bacteria,  122 
pathogenic  effects,  259 
Pus,  collection  of,  271 
Pyrogallic'acid  solution,  189 

RABBIT  cages,  264 

scales,  261 
Raising   virulence   of   organisms, 

284 

Ramsden's  micrometer,  63 
Range  of  temperature,  241 
Rat  cages,  264 

Reaction  of  medium,  effect  of,  248 
optimum,  248 
range  of,  248 

Reducing  agent  production,  232 
Reduction  of  nitrates,  232 
Reichert's  thermo-regulator,    175 
Relation  of  bacteria  to  environ- 
ment, 238 
Removal  of  material  from  culture 

tubes,  69 

Rennet  enzymes,  224 
Reproduction  of  bacteria,  116 
Resistance  to  lethal  agents,  249 
Roll  cultures,  199 
Roux's  anaerobic  culture  method, 
188 

SABOURAUD'S  medium,  170 
Saccharomyces,  morphology  of, 

110 

Safranine,  84 
Saprogenic  bacteria,  111 
Sarcina,  111 
Scales,  decimal,  261 

trap,  146 

Scalpels,  to  sterilise,  37 
Scheme  of  study  for  bacteria,  205 
Schizomycetes,  morphology  of,  111 
Scissors,  to  sterilise,  37 
Searing  irons,  288 
Sedimentation  tubes,  23 
Serial  cultivations,  199 
Serum  agar,  156,  157 
plate  cultures,  198 

bouillon,   156 

inspissator,  155 
Sewage,   analysis  of,   qualitative, 

335 

quantitative,  334 
Shake  cultivations,  181 
description  of,  217 
Shape  of  individual  colonies,  208 
Silicate  jelly,  1 72 
Size  of  individual  colonies,  208 
Slides,  27 


Slides,  to  clean  new,  27 

used,  28 
Smear  cultures,  179,  180 

description  of,  214 
Soap,  liquid,  267 
Soil,  analysis  of,  qualitative,  342 

quantitative,  339 
Soluble  toxins,  testing  of,  261 
Soyka's  milk-rice  medium,  167 
Spear-headed  spatula,  290 
Specific  serum,  collection  of,  253 

dilution  of,  255 
Spherical  aberration,  56 
Spirillum,  112 
rubrum,  302 
Spirochseta,  113 
Spleen  agar,  150 
Sporangium,  108 
Spore  formation,  arthrogenous,  11? 
endogenous,   117 
method  of,  116 
observation  of,  219 
germination,  method  of,  119 

observation  of,  219 
to  stain,  91 

Spores,  characters  of,  118 
Stab  cultures,  180 

description  of,  215 
Stage  micrometer,  60 

of  microscope,  54 
Staining  paraffin  sections,  103 

reactions  of  bacteria,  221 
Stains,  77 
Standardising  bouillon,  133 

media,  132 

Standard  soda  solution,  132 
Staphylococcus,  112 
pyogenes  albus,  296 
aureus,  296 

in  milk,  353 
citreus,  296 
Steam  steriliser,  Arnold's,  39 

Koch's,  39 
streaming,  39 
Sterigma,  108 

Sterilisation  by  chemicals,  33 
by  dry  heat,  34 
by  filters,  43 
by  moist  heat,  37 
by  streaming  steam,  39 
by  superheated  steam,    40 
of  gases,  43 
of  liquids,  44 
Sterilising  agents,  32 
Store  boxes  for  media,  140 
Streak  cultures,  179,  180 

description  of,  214 
Streaming  movement,  74- 
Streptobacillus,  112 


INDEX. 


371 


Streptococci  in  water,  332 
Streptococcus,  111 

brevis,  298 

of  bovine  mastitis,  298 

pyogenes  longus,  298 

in  milk,  353 
Streptothrix,  113 

actinomycotica,  312 
Structure  of  individual  colonies, 

211 

Subcutaneous  inoculation,  275 
Substage  condenser,  55 
Sugar  agar,  151 

bouillon,  142 

gelatine,  148 
Sulphindigotate  agar,  152 

bouillon,  143 

gelatine,  148 

Sulphuretted  hydrogen,  234 
Sunlight,  direct,  action  of,  251 
Superheated  steam,  41 
Superior  lethal  coefficient,  359 
Suppuration,  organisms  of,  295 
Surface  of  individual  colonies,  210 
Swarm  spores,  108 
Syringe  for  subcutaneous  inocula- 
tion of  solid  material,  276 

hypodermic,  265 

TATIN'S  operating  table,  272 
Taxonomy,  263,  273 
Temperature,  action  of,  241 
optimum,  241 
range,  241 
Testing  filters,  358 
Test  objects  for  objectives,  58 
Test-tubes,  17 

to  clean  infected,  25 
to  clean  new,  24 
to  plug,  29 
to  sterilise,  36 
Tetrad,  111 

Thermal  death  -  point,  J_22 
determination  of,  242 
of  spores,  244 
of  vegetative  forms,  242 
Thermophilic  bacteria,  122 
Thermo-regulators,  capsule,  175 

Reichert's,  175 
Thionine  blue,  83 
Thiothrix,  113 
Timothy  grass  bacillus,  306 
Tinned  meat,  analysis  of,  356 
Tissues  for  sectioning,  fixing,  98 
freezing,  100 
hardening,  98 
imbedding,  101 
washing,  99 
Titration  of  media,  129 


Torulae,  110 
Toxins,  testing  of,  260 
Triple  nosepiece,  58 
True  motility,  74 
Tube  cultures,  177 

inoculating,  178 

preparation  of,  178 
Tubercle  bacillus,  306 

to  stain,  95,  106 
Tubing  media,  138 
Turnip  medium,  164 

URINE  agar,  159 

gelatine,  158,  159 

media,  158 
Uschinsky's  solution,  171 

VAN  ERMENGEM'S  flagella  stain, 

89 

Vesuvin,  84 
Vibrio,  113 

cholerae,  302 
in  water,  332 

Metschnikovi,  302 

of  Kinkier  and  Prior,  302 
Virulence,  attenuating,  285 

raising,  284 

Voges'  guinea-pig  holder,  272 
Volatile  oils  as  disinfectants,  33 

WARM  stage,  58 

Washing  tissues,  99 

Water,    analysis    of,    qualitative, 

323 
quantitative,  316 

steriliser,  38 
Weighing  animals,  261 
Wertheimer's  serum  agar,  157 
Wheat  broth,  167 
Whey  agar,  162 

gelatine,  161 
Widal's  reaction,  253 
Wine  must,  167 
Winogradsky's  jelly,  172 

solutions,  172 

Wire  baskets  for  test-tubes,  36 
Wort  agar,  166 

gelatine,  166 
Wright's  anaerobic  method,  190 

XEROSIS  bacillus,  302 

YEASTS,  examination  of,  110 
Yeast  water,  170 

ZiEHL-Neelsen    staining    method, 

95,  106 
Zooglcea,  114 
Zymogenic  bacteria,  1 1 1 


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branches  ;  with  over  IOO  new  and  elaborate  tables  and  many  handsome 
illustrations.  By  W.  A.  NEWMAN  DORLAND,  M.D.,  Editor  of  "  The 
American  Pocket  Medical  Dictionary."  Large  octavo,  935  pages, 
bound  in  full  flexible  leather.  Price,  $4.50  net ;  with  thumb  index, 
$5.00  net. 

IT  DEFINES  ALL  THE  NEW  WORDS— IT  IS  UP  TO  DATE 

Borland's  Dictionary  defines  hundreds  of  the  newest  terms  not  defined  in  any 
other  dictionary — bar  none.  These  new  terms  are  live,  active  words,  taken 
right  from  modern  medical  literature. 

It  gives  the  capitalization  and  pronunciation  of  all  words.  It  makes  a  feature 
of  the  derivation  or  etymology  of  the  words.  In  some  dictionaries  the  etymology 
occupies  only  a  secondary  place,  in  many  cases  no  derivation  being  given  at  all. 
In  "  Borland,"  practically  every  word  is  given  its  derivation. 

In  "Borland"  every  word  has  a  separate  paragraph,  thus  making  it  easy  to 
find  a  word  quickly. 

The  tables  of  arteries,  muscles,  nerves,  veins  etc.,  are  of  the  greatest  help 
in  assembling  anatomic  facts.  In  them  are  classified  for  quick  study  all  the 
necessary  information  about  the  various  structures. 

In  "Borland"  every  word  is  given  its  definition — a  definition  that  defines 
in  the  fewest  possible  words.  In  some  dictionaries  hundreds  of  words  are  not 
defined  at  all,  referring  the  reader  to  some  other  source  for  the  information  he 
wants  at  once. 

Howard  A.  Kelly,  M.  D.,  Johns  Hopkins  University,  Baltimore 

"  Dr.  Borland's  dictionary  is  admirable.  It  is  so  well  gotten  up  and  of  such  convenient 
size.  No  errors  have  been  found  in  my  use  of  it." 

J.  Collins  Warren,  M.  D.,  LL.D.,  F.R.C.S.  (Hon.),  Harvard  Medical  School 

"  I  regard  it  as  a  valuable  aid  to  my  medical  literary  work.  It  is  very  complete  and  o< 
convenient  size  to  handle  comfortably.  I  use  it  in  preference  to  any  other." 


NURSING. 


Nursing  in  Diseases  of  the 
Eye,  Ear,  Nose,  and  Throat 

Nursing  in  Diseases  of  the  Eye,  Ear,  Nose,  and  Throat.     By  the 

Committee  on  Nurses  of  the  Manhattan  Eye,  Ear,  and  Throat  Hospital: 
J.  EDWARD  GILES,  M.  D.,  Surgeon  in  the  Eye  Department ;  ARTHUR  B. 
DUEL,  M.  D.  (Chairman),  Surgeon  in  the  Ear  Department ;  HARMON 
SMITH,  M.  D.,  Surgeon  in  the  Throat  Department.  Assisted  by  JOHN 
R.  SHANNON,  M.  D.,  Assistant  Surgeon  in  the  Eye  Department ;  and 
JOHN  R.  PAGE,  M.  D.,  Assistant  Surgeon  in  the  Ear  Department.  With 
chapters  by  HERBERT  B.  WILCOX,  M.  D.,  Attending  Physician  to  the 
Hospital;  and  Miss  EUGENIA  D.  AYERS,  Superintendent  of  Nurses. 
I2mo  of  260  pages,  illustrated.  Cloth,  $1.50  net. 

A  VALUABLE  BOOK 

This  is  a  practical  book,  prepared  by  surgeons  who,  from  their  experience  in 
the  operating  amphitheater  and  at  the  bedside,  have  realized  the  shortcomings  of 
present  nursing  books  in  regard  to  eye,  ear,  nose,  and  throat  nursing.  The  scope 
of  the  work  has  been  limited  to  what  an  intelligent  nurse  should  know,  and  the 
style  throughout  is  simple,  plain,  and  definite. 

New  York  Medical  Journal 

"  Every  side  of  the  question  has  been  fully  taken  into  consideration." 

Stoney's 
Materia  Medica  for  Nurses 

Practical  Materia  Medica  for  Nurses,  with  an  Appendix  containing 
Poisons  and  their  Antidotes,  with  Poison-Emergencies  ;  Mineral  Waters ; 
Weights  and  Measures ;  Dose-List,  and  a  Glossary  of  the  Terms  used 
in  Materia  Medica  and  Therapeutics.  By  EMILY  A.  M.  STONEY,  of  the 
Carney  Hospital,  South  Boston.  I2mo  of  3OOpages.  Cloth,  $1.50  net. 

THE    NEW   (3d)    EDITION 

In  making  the  revision  for  this  new  third  edition,  all  the  newer  drugs  have 
been  introduced  and  fully  discussed.  The  consideration  of  the  drugs  includes 
their  sources  and  composition,  their  various  preparations,  physiologic  actions, 
directions  for  administering,  and  the  symptoms  and  treatment  of  poisoning. 

Journal  of  the  American  Medical  Association 

"  So  far  as  we  can  see,  it  contains  everything  that  a  nurse  ought  to  know  in  regard  to  drugs. 
As  a  reference-book  for  nurses  it  will  without  question  be  very  useful." 


SAUNDERS*    BOOKS   ON 


Stoney's  Nursing 


Practical  Points  in  Nursing :  for  Nurses  in  Private  Practice.     By 

EMILY  A.  M.  STONEY,  Superintendent  of  the  Training  School  for  Nurses 
at  the  Carney  Hospital,  South  Boston,  Mass.  12  mo.  of  495  pages, 
fully  illustrated.  Cloth,  $1.75  net. 

THE  NEW  (4th)  EDITION 

In  this  volume  the  author  explains  the  entire  range  of  private  nursing  as  dis- 
tinguished from  hospital  nursing,  and  the  nurse  is  instructed  how  best  to  meet  the 
various  emergencies  of  medical  and  surgical  cases  when  distant  from  medical  or 
surgical  aid  or  when  thrown  on  her  own  resources.  An  especially  valuable  feature 
will  be  found  in  the  directions  how  to  improvise  everything  ordinarily  needed  in  the 
sick-room. 

The  Lancet,  London 

"A  very  complete  exposition  of  practical  nursing  in  its  various  branches,  including  obstetric 
and  gynecologic  nursing.     The  instructions  given  are  full  of  useful  detail." 


Stoney's  Technic  for  Nurses 

Bacteriology  and  Surgical  Technic  for  Nurses.  By  EMILY  A.  M. 
STONEY,  Superintendent  at  Carney  Hospital,  South  Boston.  Revised 
by  FREDERIC  R.  GRIFFITH,  M.  D.,  Surgeon,  of  New  York.  i2mo, 
311  pages,  illustrated.  Cloth,  $1.50  net. 

THE    NEW    (3d)     EDITION 
Trained  Nurse  and  Hospital  Review 

"  These  subjects  are  treated  most  accurately  and  up  to  date,  without  the  superfluous  reading 
which  is  so  often  employed.  .  .  .  Nurses  will  find  this  book  of  the  greatest  value  both  during 
their  hospital  course  and  in  private  practice." 

Spratling  on  Epilepsy 

Epilepsy  and  Its  Treatment.  By  WILLIAM  P.  SPRATLING,  M.  D., 
Medical  Superintendent  of  the  Craig  Colony  for  Epileptics,  Sonyea, 
New  York.  Octavo  of  522  pages,  fully  illustrated.  Cloth,  $4.00  net. 

The  Lancet.  London 

"  Dr.  Sp ratling's  work  is  written  throughout  in  a  clear  and  readable  style.  .  .  .  The  work 
is  a  mine  of  information  on  the  whole  subject  of  epilepsy  and  its  treatment." 


NURSING. 


Aikens'  Primary  Studies  for  Nurses  illustrated 

PRIMARY  STUDIES  FOR  NURSES:  A  Text-Book  for  First-year  Pupil 
Nurses.  By  CHARLOTTE  A.  AIKENS,  formerly  Director  of  Sibley  Memorial 
Hospital,  Washington,  D.  C.  i2mo  of  450  pages,  illus.  Cloth,  $1.75  net. 

This  work  brings  together  in  concise  form  well-rounded  courses  of  lessons 
in  all  subjects  which,  with  practical  nursing  technic,  constitute  the  primary 
studies  in  a  nursing  course. 

Trained  Nurse  and  Hospital  Review 

"It  is  safe  to  say  that  any  pupil  who  has  mastered  even  the  major  portion  of  this  work 
would  be  one  of  the  best  prepared  first-year  pupils  that  ever  stood  for  examination." 

Aikens'  Clinical  Studies  for  Nurses  fSS" 

CLINICAL  STUDIES  FOR  NURSES.  By  CHARLOTTE  A.  AIKENS,  formerly 
Director  of  Sibley  Memorial  Hospital,  Washington,  D.  C.  i2mo  of 
510  pages,  illustrated.  Cloth,  $2.00  net. 

This  new  work  is  written  along  the  same  lines  as  Miss  Aikens'  former 
work  on  "Primary  Studies,"  to  which  it  is  a  companion  volume.  It  takes 
up  all  subjects  taught  during  the  second  and  third  years  and  takes  them 
up  in  a  concise,  forceful  way. 

Dietetic  and  Hygienic  Gazette 

"  There  is  a  large  amount  of  practical  information  in  this  book  which  the  experienced 
nurse,  as  well  as  the  undergraduate,  will  consult  with  profit.  The  illustrations  are 
numerous  and  well  selected." 

Aikens'  Training-School  Methods 

HOSPITAL  TRAINING-SCHOOL  METHODS  AND  THE  HEAD  NURSE.  By 
CHARLOTTE  A.  AIKENS,  formerly  Director  of  Sibley  Memorial  Hospital, 
Washington,  D.  C.  12 mo  of  267  pages.  Cloth,  $1.50  net. 

Trained  Nurse  and  Hospital  Review 

"  There  is  not  a  chapter  in  the  book  that  does  not  contain  valuable  suggestions." 

Aikens9  Hospital  Management  J"*t  Ready 

HOSPITAL  MANAGEMENT.  BY  CHARLOTTE  A.  AIKENS,  formerly  Direc- 
tor of  Sibley  Memorial  Hospital,  Washington,  D.  C.  i2mo  of  488 
pages,  illustrated.  Cloth,  $3.00  net. 

Miss  Aikens'  long  experience  as  hospital  director  has  well  fitted  her  to 
write  on  this  subject.  Her  book  is  a  concise,  careful,  and  thoughtful  discus- 
sion of  the  subject,  presented  in  a  way  that  must  strike  home  at  once. 


SAUN&ERS*    BOOKS    ON 


Hoxie's  Medicine  for  Nurses 

Practice  of  Medicine  for  Nurses.  A  Text-Book  for  Nurses  and  Students, 
of  Domestic  Science,  and  a  Hand-Book  for  All  Those  Who  Care  for  the  Sicfc^ 
By  GEORGE  HOWARD  HOXIE,  M.  D.,  Professor  of  Internal  Medicine,  U»i-- 
versity  of  Kansas.  With  a  Chapter  on  Technic  of  Nursing  by  PEARL  L.. 
LAPTAD,  Principal  of  the  Training  School  for  Nurses,  University  of  Kansas,. 
I2mo  of  284  pages,  illustrated.  Cloth,  $1.50  net, 

This  work  is  truly  a  practice  of  medicine  for  the  nurse,  enabling  her  to  recognize  any 
signs  and  changes  that  may  occur  between  visits  of  the  physician,  and,  if  necessary,  to, 
combat  them  until  the  physician's  arrival.  This  information  the  author  presents  in  a.  way- 
most  acceptable,  particularly  emphasizing  the  nurse's  part. 

Trained  Nurse  and  Hospital  Review 

"  This  book  has  our  unqualified  approval." 


McCombs'  Diseases  of  Children  for  Nurses  New 

Diseases  of  Children  for  Nurses.  By  ROBERT  S.  McCoMBS,  M.  D.,, 
Instructor  of  Nurses  at  the  Children's  Hospital  of  Philadelphia.  I2mo  of 
470  pages,  illustrated.  Cloth,  $2.00  net. 

Dr.  McCombs'  experience  in  lecturing  to  nurses  has  enabled  him  to  emphasize  just  those- 
points  that  nurses  most  need  to  know.  The  nursing  side  has  been  written  by  head  nurses,, 
especially  praiseworthy  being  the  work  of  Miss  Jennie  Manly. 

National  Hospital  Record 

"  We  have  needed  a  good  work  on  children's  diseases  adapted  for  nurses'  use,  and  this, 
volume  admirably  fills  the  want." 

Wilson's   Obstetric  Nursing 

A  Reference  Hand-Book  of  Obstetric  Nursing.  By  W.  REYNOLDS 
WILSON,  M.  D.,  Visiting  Physician  to  the  Philadelphia  Lying-in  Charity. 
32mo  of  258  pages,  illustrated.  Flexible  leather,  $1.25  net. 

Dr.  Wilson's  work  discusses  the  subject  of  obstetrics  entirely  from  the  nurse's  point  o| 
view,  presenting  in  detail  everything  connected  with  pregnancy  and  labor  and  their  man- 
agement. The  text  is  copiously  illustrated. 

American  Journal  of  Obstetrics 

"  Every  page  emphasizes  the  nurse's  relation  to  the  case." 

Friihwald  and  Westcott  on  Children 

Diseases  of  Children.  A  Practical  Reference  Book  for  Students  and 
Practitioners.  By  PROFESSOR  DR.  FERDINAND  FRUHWALD,  of  Vienna. 
Edited,  with  additions,  by  THOMPSON  S.  WESTCOTT,  M.  D.,  University  of 
Pennsylvania.  Octavo,  533  pages,  176  illustrations.  Qoth,  $4.50  net. 

Boyd's  State  Registration  for  Nurses 

State  Registration  for  Nurses.  By  LOUIE  CROFT  BOYD*  R,  N.,  Graduate 
Colorado  Training-school  for  Nurses.  Octavo  of  42  pages*  50  cents  net. 


NURSING. 


Macfarlane's  Gynecology  for  Nurses  illustrated 

A  REFERENCE  HAND-BOOK  OF  GYNECOLOGY  FOR  NURSES.  By  CATH- 
ARINE MACFARLANE,  M.  D.,  Gynecologist  to  the  Woman's  Hospital  of 
Philadelphia.  321110  of  150  pages,  with  70  illustrations.  Flexible 
leather,  $1.25  net. 

A.  M.  Seabrook,   M.  D.,    Woman's  Medical  College  of  Philadelphia. 

"  It  is  a  most  admirable  little  book,  covering  in  a  concise  but  attractive  way  the  subject  from 
the  nurse's  standpoint." 

Galbraith's  Personal  Hygiene  and  Physical  Training 

for  Women  Recently  Issued 

PERSONAL  HYGIENE  AND  PHYSICAL  TRAINING  FOR  WOMEN.  By 
ANNA  M.  GALBRAITH,  M.D.,  Fellow  New  York  Academy  of  Medicine,, 
i2mo  of  371  pages,  with  original  illustrations.  Cloth,  $2.00  net. 

Dr.  Galbraith's  book  is  just  what  has  long  been  needed — a  simple  manual 
of  hygiene  and  physical  training  along  scientific  lines. 

De  Lee's   Obstetrics  for  Nurses  New  (3d)  Editfon 

OBSTETRICS  FOR  NURSES.  By  JOSEPH  B.  DELEE,  M.  D.,  Professor  of 
Obstetrics  in  the  Northwestern  University  Medical  School.  12010  vol- 
ume of  512  pages,  fully  illustrated.  Cloth,  $2.50  net. 

J.  Clifton    Edgar,  M.  D., 

Professor  of  Obstetrics  and  Clinical  Midwifery,  Cornell  Medical  School y  N.  Y. 

"  It  is  far-and-away  the  best  that  has  come  to  my  notice,  and  I  shall  take  great  pleasure  in  recom- 
mending it  to  my  nurses  and  students  as  well." 

Davis'  Obstetric  Nursing  New  (3d) 

OBSTETRIC  AND  GYNECOLOGIC  NURSING.  By  EDWARD  P.  DAVIS,  A.  M., 
M.  D.,  Professor  of  Obstetrics,  Jefferson  Medical  College  and  Philadel- 
phia Polyclinic.  i2mo  of  436  pages,  illustrated.  Buckram,  $1.75  net. 

The  Lancet,  London 

"  Not  only  nurses,  but  even  newly  qualified  medical  men,  would  learn  a  great  deal  by  a  perusal  of 
this  book.  It  is  written  in  a  clear  and  pleasant  style,  and  is  a  work  we  can  recommend." 

Beck's   Hand-Book  for  Nurses  New  (2d)  Editioll 

A  REFERENCE  HAND-BOOK  FOR  NURSES.  By  AMANDA  K.  BECK,  of 
Chicago,  111.  32mo  of  200  pages.  Flexible  leather,  $1.25  net. 

This  little  book  contains  information  upon  every  question  that  comes  to  a 
nurse  in  her  daily  work,  and  embraces  all  the  information  that  she  requires 
to  carry  out  any  directions  given  by  the  physician. 

Boston  Medical  and  Surgical  Journal 

"  Muflt  be  regarded  as  an  extremely  useful  book,  not  only  for  nurses,  but  for  physicians." 


10 


SAUNDERS*    BOOKS   ON 


Register's  Fever  Nursing 

A  TEXT-BOOK  ON  PRACTICAL  FEVER  NURSING.  By  EDWARD  C. 
B.EGISTER,  M.  D.,  Professor  of  the  Practice  of  Medicine  in  the  North 
Carolina  Medical  College.  i2mo  of  352  pages.  Cloth,  $2.50  net. 

The  work  completely  covers  the  field  of  practical  fever  nursing.  The  illustrations  shov» 
the  nurse  how  to  perform  those  measures  that  come  within  her  province. 

Trained  Nurse  and  Hospital  Review 

"  Nurses  will  find  this  book  of  great  value  in  this  practical  branch  of  their  work." 

Hecker,  Trumpp,  and  Abt  on  Children 

ATLAS  AND  EPITOME  OF  DISEASES  OF  CHILDREN.  By  Dr.  R.  HECKER 
and  Dr.  J.  TRUMPP,  of  Munich.  Edited,  with  additions,  by  ISAAC  A. 
ABT,  M.D.,  Assistant  Professor  of  Diseases  of  Children,  Rush  Medical 
College,  Chicago.  With  48  colored  plates,  144  text-cuts,  and  453  pages 
of  text.  Cloth,  15.00  net. 

The  many  excellent  lithographic  plates  represent  cases  seen  in  the  authors'  clinics,  and 
have  been  selected  with  great  care,  keeping  constantly  in  mind  the  practical  needs  of  the 
general  practitioner.  These  beautiful  pictures  are  so  true  to  nature  that  their  study  is 
equivalent  to  actual  clinical  observation.  The  editor,  Dr.  Isaac  A.  Abt,  has  added  all  new 
methods  of  treatment. 

Johns  Hopkins  Hospital  Bulletin 

"  The  entire  field  has  been  covered.  With  the  excellent  plates,  it  will  be  found  of  real 
value  to  both  students  and  practitioners." 

Lewis*  Anatomy  and   Physiology         The  New  (id)  Edition 

ANATOMY  AND  PHYSIOLOGY  FOR  NURSES.  By  LEROY  LEWIS,  M.D., 
Surgeon  to  and  Lecturer  on  Anatomy  and  Physiology  for  Nurses  at  the 
Lewis  Hospital,  Bay  City,  Michigan.  i2mo  of  375  pages,  with  150 
illustrations.  Cloth,  $1.75  net. 

A  demand  for  such  a  work  as  this,  treating  the  subjects  from  the  nurses  point  of  view, 
has  long  existed.  Dr.  Lewis  has  based  the  plan  and  scope  of  this  work  on  the  methods 
employed  by  him  in  teaching  these  branches,  making  the  text  unusually  simple  and  clear. 

The  Nurses  Journal  of  the  Pacific  Coast 

"  It  is  not  in  any  sense  rudimentary,  but  comprehensive  in  its  treatment  of  the  subjects 
in  hand.  The  application  of  the  knowledge  of  anatomy  in  the  care  of  the  patient  is 
emphasized." 

Friedenwald  and  Ruhrah's  Dietetics  New  (id)  Edition 

DIETETICS  FOR  NURSES.  By  JULIUS  FRIEDENWALD,  M.  D.,  Professor 
of  Diseases  of  the  Stomach,  and  JOHN  RUHRAH,  M.  D.,  Professor  of 
Diseases  of  Children,  College  of  Physicians  and  Surgeons,  Baltimore. 
i2mo  volume  of  395  pages.  Cloth,  $1.50  net. 

This  work  has  been  prepared  to  meet  the  needs  of  the  nurse,  both  in  the  training 
school  and  after  graduation.  It  aims  to  give  the  essentials  of  dietetics,  considering  briefly 
the  physiology  of  digestion  and  the  various  classes  of  foods  and  the  part  they  play  in 
nutrition. 

American  Journal  of  Nursing 

"  It  is  exactly  the  book  for  which  nurses  and  others  have  long  and  vainly  sought.  A 
simple  manual  of  -dietetics,  which  does  not  turn  into  a  cook-book  at  the  end  of  the  first 
or  second  chapter. 


NURSING  AND  CHILDREN.  II 


T»        i»       T>  mr          • 

Pauls  Fever  Nursing  New  (2d)  Edition 

NURSING  IN  THE  ACUTE  INFECTIOUS  FEVERS.  By  GEORGE  P.  PAUL, 
M.D.,  Assistant  Visiting  Physician  to  the  Samaritan  Hospital,  Troy,  N.  Y. 
i2mo  of  246  pages.  Cloth,  $1.00  net. 

Dr.  Paul  has  taken  great  pains  in  the  presentation  of  the  care  and  management  of  each 
fever.  The  book  treats  of  fevers  in  general,  then  each  fever  is  discussed  individually,  and 
the  latter  part  of  the  book  deals  with  practical  procedures  and  valuable  information. 

The  London  Lancet 

"  The  book  is  an  excellent  one  and  will  be  of  value  to  those  for  whom  it  is  intended. 
It  is  well  arranged,  the  text  is  clear  and  full,  and  the  illustrations  are  good." 

Paul's  Materia  Medica  for  Nurses 

MATERIA  MEDICA  FOR  NURSES.  By  GEORGE  P.  PAUL,  M.D.,  Assistant 
Visiting  Physician  to  the  Samaritan  Hospital,  Troy.  i2mo  of  240  pages, 
Cloth,  #1.50  net. 

Dr.  Paul  arranges  the  physiologic  actions  of  the  drugs  according  to  the  action  of  the 
drug  and  not  the  organ  acted  upon.  An  important  section  is  that  on  pretoxic  signs, 
giving  the  warnings  of  the  full  action  or  the  beginning  toxic  effects  of  the  drug,  which, 
if  heeded,  may  prevent  many  cases  of  drug  poisoning. 

The  Medical  Record,  New  York 

"This  volume  will  be  of  real  help  to  nurses;  the  material  is  well  selected  and  well 
arranged,  and  the  book  is  as  readable  as  it  is  useful." 

Pyle's  Personal  Hygiene  The  New  (4th)  Edition 

A  MANUAL  OF  PERSONAL  HYGIENE  :  Proper  Living  upon  a  Physiologic 
Basis.  By  Eminent  Specialists.  Edited  by  WALTER  L.  PYLE,  A.M., 
M.D.,  Assistant  Surgeon  to  Wills  Eye  Hospital,  Philadelphia.  Octavo 
volume  of  472  pages,  fully  illustrated.  Cloth,  £1.50  net. 

To  this  new  edition  there  have  been  added,  and  fully  illustrated,  chapters  on  Domestic 
Hygiene  and  Home  Gymnastics,  besides  an  appendix  containing  methods  of  Hydro- 
therapy,  Mechanotherapy,  and  First  Aid  Measures.  There  is  also  a  Glossary  of  the 
medical  terms  used. 

Boston  Medical  and  Surgical  Journal 

"  The  work  has  been  excellently  done,  there  is  no  undue  repetition,  and  the  writers 
have  succeeded  unusually  well  in  presenting  facts  of  practical  significance  based  on  sound 
knowledge." 

Galbraith's  Four  Epochs  of  Woman's  Life     second  Edition 

THE   FOUR   EPOCHS  OF  WOMAN'S   LIFE.     By  ANNA  M.  GALBRAITH, 
M.D.      With  an  Introductory  Note  by  JOHN  H.  MUSSER,  M.D.,  Univer- 
sity of  Pennsylvania.      i2mo  of  247  pages.     Cloth,  $1.50  net. 
Birmingham  Medical  Review 

"  We  do  not  as  a  rule  care  for  medical  books  written  for  the  instruction  of  the  public  ; 
but  we  must  admit  that  the  advice  in  Dr.  Galbraith's  work  is  in  the  main  wise  and  whole- 


Starr  on  Children  second  Edition 

AMERICAN  TEXT-BOOK  OF  DISEASES  OF  CHILDREN.  Edited  by  Louis 
STARR,  M.D.,  assisted  by  THOMPSON  S.  WESTCOTT,  M.D.  Octavo,,  1244 
pages,  illustrated.  Cloth,  $7.00  net;  Half  Morocco,  #8.50  net. 


12  SAUNDERS*    BOOKS   ON 

Brower  and  Bannister 
on  Insanity 

A  Practical  Manual  of  Insanity.  For  the  Student  and  General 
Practitioner.  By  DANIEL  R.  BROWER,  A.M.,  M.D.,  LL.  D.,  Professor 
of  Nervous  and  Mental  Diseases  in  Rush  Medical  College,  in  affiliation 
with  the  University  of  Chicago  ;  and  HENRY  M.  BANNISTER,  A.  M., 
M.  D.,  formerly  Senior  Assistant  Physician,  Illinois  Eastern  Hospital 
for  the  Insane.  'Handsome  octavo  of  426  pages,  with  a  number  of 
full-page  inserts.  Cloth,  $3.00  net. 

FOR  STUDENT  AND  PRACTITIONER 

This  work,  intended  for  the  student  and  general  practitioner,  is  an  intelligible, 
up-to-date  exposition  of  the  leading  facts  of  psychiatry,  and  will  be  found  of  in- 
valuable service,  especially  to  the  busy  practitioner  unable  to  yield  the  time  for  a 
more  exhaustive  study.  The  work  has  been  rendered  more  practical  by  omitting 
elaborate  case  records  and  pathologic  details,  as  well  as  discussions  of  speculative 
and  controversial  questions. 

American  Medicine 

"  Commends  itself  for  lucid  expression  in  clear-cut  English,  so  essential  to  the  student  in 
any  department  of  medicine.  .  .  .  Treatment  is  one  of  the  best  features  of  the  book,  and  for 
this  aspect  is  especially  commended  to  general  practitioners." 

Bergey's  Hygiene 

The  Principles  of  Hygiene:  A  Practical  Manual  for  Students, 
Physicians,  and  Health  Officers.  By  D.  H.  BERGEY,  A.  M.,  M.  D., 
Assistant  Professor  of  Bacteriology  in  the  University  of  Pennsylvania. 
Octavo  volume  of  555  pages,  illustrated.  Cloth,  $3.00  net. 

THE  NEW  (3d)  EDITION 

This  book  is  intended  to  meet  the  needs  of  students  of  medicine  in  the 
acquirement  of  a  knowledge  of  those  principles  upon  which  modern  hygienic 
practises  are  based,  and  to  aid  physicians  and  health  officers  in  familiarizing 
themselves  with  the  advances  made  in  hygiene  and  sanitation  in  recent  years. 
This  new  third  edition  has  been  very  carefully  revised,  and  much  new  matter 
added,  so  as  to  include  the  most  recent  advancements. 

Buffalo  Medical  Journal 

"  It  will  be  found  of  value  to  the  practitioner  of  medicine  and  the  practical  sanitarian  ;  and 
students  of  architecture,  who  need  to  consider  problems  of  heating,  lighting,  ventilation,  water 
supply,  and  sewage  disposal,  may  consult  it  with  profit." 


CHILDREN  AND  HYGIENE.  13 

Griffith's  Care  of  the  Baby 

The  Care  of  the  Baby.  By  J.  P.  CROZER  GRIFFITH,  M.  D.,  Clinical 
Professor  of  Diseases  of  Children,  University  of  Penn. ;  Physician  to  the 
Children's  Hospital,  Phila.  I2mo,  455  pp.  Illustrated.  Cloth,  $1.50  net. 

THE  NEW  (5th)  EDITION 

The  author  has  endeavored  to  furnish  a  reliable  guide  for  mothers.  He  has 
made  his  statements  plain  and  easily  understood,  in  the  hope  that  the  volume 
may  be  of  service  not  only  to  mothers  and  nurses,  but  also  to  students  and  practi- 
tioners whose  opportunities  for  observing  children  have  been  limited. 

New  York  Medical  Journal 

"  We  are  confident  if  this  little  work  could  find  its  way  into  the  hands  of  every  trained 
nurse  and  of  every  mother,  infant  mortality  would  be  lessened  by  at  least  fifty  per  cent." 

Crothers*  Morphinism 

Morphinism  and  Narcomania  from  Opium,  Cocain,  Ether,  Chloral, 
Chloroform,  and  other  Narcotic  Drugs ;  also  the  Etiology,  Treatment, 
and  Medicolegal  Relations.  By  T.  D.  CROTHERS,  M.  D.,  Superintendent 
of  Walnut  Lodge  Hospital,  Hartford,  Conn.  Handsome  I2mo  of  351 
pages.  Cloth,  $2.00  net. 

The  Lancet,  London 

"An  excellent  account  of  the  various  causes,  symptoms,  and  stages  of  morphinism,  the 
discussion  being  throughout  illuminated  by  an  abundance  of  facts  of  clinical,  psychological,  and 
social  interest." 

Ruhrah's   Diseases   of   Children 

A  Manual  of  Diseases  of  Children.  By  JOHN  RUHRAH,  M.  D., 
Professor  of  Diseases  of  Children,  College  of  Physicians  and  Surgeons, 
Baltimore.  I2mo  of  534  pages,  fully  illustrated.  Flexible  leather] 
$2.50  net. 

THE  NEW  (3d)  EDITION 

In  revising  this  work  for  the  second  edition  Dr.  Ruhrah  has  carefully  in- 
corporated all  the  latest  knowledge  on  the  subject.  All  the  important  facts  are 
given  concisely  and  explicitly,  the  therapeutics  of  infancy  and  childhood  being 
outlined  very  carefully  and  clearly.  There  are  also  directions  for  dosage  and 
prescribing,  and  many  useful  prescriptions  are  included. 

American  Journal  of  the  Medical  Sciences 

"Treatment  has  been  satisfactorily  covered,  being  quite  in  accord  with  the  best  teaching, 
yet  withal  broadly  general  and  free  from  stock  prescriptions." 


14  SAUNDERS'    BOOKS   ON 

Peterson  and  Haines' 
Legal  Medicine  &  Toxicology 


A  Text-Book  of  Legal  Medicine  and  Toxicology.  Edited  by 
FREDERICK  PETERSON,  M.  D.,  Professor  of  Psychiatry  in  the  College 
of  Physicians  and  Surgeons,  New  York;  and  WALTER  S.  HAINES, 
M.  D.,  Professor  of  Chemistry,  Pharmacy,  and  Toxicology,  Rush 
Medical  College,  in  affiliation  with  the  University  of  Chicago.  Two- 
imperial  octavo  volumes  of  about  750  pages  each,  fully  illustrated. 
Per  volume:  Cloth,  $5.00  net;  Sheep  or  Half  Morocco,  $6.50  net 
Sold  by  Subscription. 

IN  TWO  VOLUMES 

The  object  of  the  present  work  is  to  give  to  the  medical  and  legal  professions 
a  comprehensive  survey  of  forensic  medicine  and  toxicology  in  moderate  compass. 
This,  it  is  believed,  has  not  been  done  in  any  other  recent  work  in  English.  Under 
"  Expert  Evidence"  not  only  is  advice  given  to  medical  experts,  but  suggestions 
are  also  made  to  attorneys  as  to  the  best  methods  of  obtaining  the  desired  infor- 
mation from  the  witness.  An  interesting  and  important  chapter  is  that  on  "The 
Destruction  and  Attempted  Destruction  of  the  Human  Body  by  Fire  and  Chemi- 
cals." A  chapter  not  usually  found  in  works  on  legal  medicine  is  that  on  "  The 
Medicolegal  Relations  of  the  X-Rays." 
Columbia  Law  Review 

"  For  practitioners  in  criminal  law  and  for  those  in  medicine  who  are  called  upon  to  give 
court  testimony  in  all  its  various  forms  ...  it  is  extremely  valuable." 


Fiske's  Human  Body 

Structure  and  Functions  of  the  Body.  By  ANNETTE  FISKE,  A.M.,. 
Graduate  of  the  Waltham  Training  School  for  Nurses.  I2mo  of  221 
pages,  illustrated.  Cloth,  $1.25  net. 

JUST  READY 

The  way  in  which  this  book  presents  anatomy  and  physiology  is  a  departure 
from  the  usual  method — a  departure,  however,  of  a  very  practical  kind.  Miss 
Fiske  has  woven  the  physiology  in  with  the  anatomy,  thus  making  her  work  a 
most  readable  one.  It  is  an  extremely  practical  book — one  that  can  be  readily 
understood. 


LEGAL  MEDICINE. 


Draper's  Legal  Medicine 

A  Text-Book  of  Legal  Medicine.  By  FRANK  WINTHROP  DRAPER,  A.  M., 
M.  D.,  Late  Professor  of  Legal  Medicine  in  Harvard  University,  Boston. 
Octavo  of  573  pages,  illustrated.  Cloth,  $4.00  net  ;  Half  Morocco,  $5.50  net. 

Hon.  Olin  Bryan,  LL.  B.,  Baltimore  Medical  College. 

"  A  careful  reading  of  Draper's  Legal  Medicine  convinces  me  of  the  excellent  character 
of  the  work.  It  is  comprehensive,  thorough,  and  must,  of  a  necessity,  prove  a  splendid 
acquisition  to  the  libraries  of  those  who  arc  interested  in  medical  jurisprudence." 

Chapman's  Medical  Jurisprudence  Third  Edition 

Medical  Jurisprudence,  Insanity,  and  Toxicology.  By  HENRY  C. 
CHAPMAN,  M.  D.,  late  Professor  of  Institutes  of  Medicine  and  Medical  Juris- 
prudence in  Jefferson  Medical  College,  Philadelphia.  I2mo  of  329  pages, 
illustrated.  Cloth,  $1.75  net. 

Golebiewski  and  Bailey's  Accident  Diseases 

Atlas  and  Epitome  of  Diseases  Caused  by  Accidents.     By  DR.  ED. 

GOLEBIEWSKI,  of  Berlin.  Edited,  with  additions,  by  PEARCE  BAILEY,  M.  D., 
Consulting  Neurologist  to  St.  Luke's  Hospital,  New  York.  With  71  colored 
illustrations  on  40  plates,  143  text  illustrations,  and  549  pages  of  text.  Cloth, 
$4.00  net.  In  Saunders1  Hand-Atlas  Series. 

Hofmann  and  Peterson's   Legal   Medicine 


Atlas  of  Legal  Medicine.  By  DR.  E.  VON  HOFMANN,  of  Vienna. 
Edited  by  FREDERICK  PETERSON,  M.  D.,  Professor  of  Psychiatry  in  the 
College  of  Physicians  and  Surgeons,  New  York.  With  120  colored  figures 
on  56  plates  and  193  half-tone  illustrations.  Cloth,  $3.50  net. 

Jakob  and  Fisher's  Nervous  System 

and     itS     Diseases  In  Saunders*  Hand-Atlases 

Atlas  and   Epitome  of  the  Nervous  System  and  its  Diseases.     By 

PROFESSOR  DR.  CHR.  JAKOB,  of  Erlangen.  From  the  Second  Revised 
German  Edition.  Edited,  with  additions,  by  EDWARD  D.  FISHER,  M.  D.,. 
Professor  of  Diseases  of  the  Nervous  System,  University  and  Bellevue 
Hospital  Medical  College,  New  York.  With  83  plates  and  copious  text. 
Cloth,  $3.  50  net. 

Abbott's  Transmissible  Diseases  second  Edition 

The  Hygiene  of  Transmissible  Diseases  :  Their  Causes,  Modes  of  Dis- 
semination, and  Methods  of  Prevention.  By  A.  C.  ABBOTT,  M.  D.,  Pro- 
fessor of  Hygiene  and  Bacteriology,  University  of  Pennsylvania.  Octavo  of 
351  pages,  illustrated.  Cloth,  12.50  net 


16  SAWDERS'  BOOKS  ON  CHILDREN. 

American  Pocket  Dictionary         just  Ready-New  (?th)  Edition 

AMERICAN  POCKET  MEDICAL  DICTIONARY.  Edited  by  W.  A.  NEW- 
MAN BORLAND,  M.  D.,  Editor  "American  Illustrated  Medical  Dic- 
tionary." Containing  the  pronunciation  and  definition  of  the  principal 
words  used  in  medicine  and  kindred  sciences,  with  64  extensive  tables. 
With  610  pages.  Flexible  leather,  with  gold  edges,  $1.00  net;  with 
patent  thumb  index,  $1.25  net. 

"  I  can  recommend  it  to  our  students  without  reserve."  —  J.  H.  HOLLAND,  M.  D.,  Dean 
9f  the  Jefferson  Medical  College,  Philadelphia. 

Morrow's  Immediate  Care  of  Injured 

IMMEDIATE  CARE  OF  THE  INJURED.  By  ALBERT  S.  MORROW,  M.  D., 
Attending  Surgeon  to  the  New  York  City  Hospital  for  the  Aged  and 
Infirm.  Octavo  of  340  pages,  with  238  illustrations.  Cloth,  $2.50  net. 

Dr.  Morrow's  book  on  emergency  procedures  is  written  in  a  definite  and  decisive  style, 
the  reader  being  told  just  what  to  do  in  every  emergency.  It  is  a  practical  book  for  every 
day  use,  and  the  large  number  of  excellent  illustrations  can  not  but  make  the  treatment  to 
be  pursued  in  any  case  clear  and  intelligible.  Physicians  and  nurses  will  find  it  indispensible. 


Powell's  Diseases  of  Children  Third  Edition, 

ESSENTIALS  OF  THE  DISEASES  OF  CHILDREN.  By  WILLIAM  M.  POWELL, 
M.  D.  Revised  by  ALFRED  HAND,  JR.,  A.  B.,  M.  D.,  Dispensary 
Physician  and  Pathologist  to  the  Children's  Hospital,  Philadelphia. 
i2mo  volume  of  259  pages.  Cloth,  $1.00  net.  In  Saunders9 
Question-  Compend  Series. 

Shaw  on  Nervous  Diseases  and  Insanity      Fourth  Edition 

ESSENTIALS  OF  NERVOUS  DISEASES  AND  INSANITY  :  Their  Symptoms 
and  Treatment.  A  Manual  for  Students  and  Practitioners.  By  the  late 
JOHN  C.  SHAW,  M.  D.,  Clinical  Professor  of  Diseases  of  the  Mind  and 
Nervous  System,  Long  Island  College  Hospital,  New  York.  i2mo  of 
204  pages,  illustrated.  Cloth,  $1.00  net.  In  Saunders'  Question-  Com- 
pend Series. 

*'  Clearly  and  intelligently  written  ;  we  have  noted  few  inaccuracies  and  several  sug- 
gestive points.  Some  affections  unmentioned  in  many  of  the  large  text-books  are  noted." 
—  Boston  Medical  and  Surgical  Journal. 

Starr's  Diets  for  Infants  and  Children 

DIETS  FOR  INFANTS  AND  CHILDREN  IN  HEALTH  AND  IN  DISEASE.  By 
Louis  STARR,  M.  D.,  Consulting  Pediatrist  to  the  Maternity  Hospital, 
Philadelphia.  230  blanks  (pocket-book  size).  Bound  in  flexible  leather, 
$  i.  25  net. 

Grafstrom's  Mechano-Therapy  second  Revised  Edition 

A  TEXT-BOOK  OF  MECHANO-THERAPY  (Massage  and  Medical  Gymnas- 
tics). By  AXEL  V.  GRAFSTROM,  B.  Sc.,  M.  D.,  Attending  Physician  to 
the  Gustavus  Adolphus  Orphange,  Jamestown,  New  York.  i2mo,  200 
pages,  illustrated.  Cloth,  £1.25  net. 


THIS  BOOK  IS  DUE  ON  THE  LAST  DATE 
STAMPED  BELOW 

AN  INITIAL  FINE  OF  25  CENTS 

WILL  BE  ASSESSED  FOR  FAILURE  TO  RETURN 
THIS  BOOK  ON  THE  DATE  DUE.  THE  PENALTY 
WILL  INCREASE  TO  SO  CENTS  ON  THE  FOURTH 
DAY  AND  TO  $1.OO  ON  THE  SEVENTH  DAY 
OVERDUE. 


BIOLOGY  LIBRARY 


OCT  1  6  1941 


/"   A    X 


1954 


' 


APR  2  6  1963 


LD  21-5m-7,'37 


T.C  88540 


